Manuals: Targeted Genotyping System User`s Guide

Manuals: Targeted Genotyping System User`s Guide
Affymetrix® GeneChip® Scanner 3000 Targeted
Genotyping System User Guide
P/N 702164 Rev. 2
For research use only.
Not for use in diagnostic procedures.
Trademarks
, GeneChip®, HuSNP®, GenFlex®, Flying Objective™, CustomExpress®,
Affymetrix®,
CustomSeq®, NetAffx™, Tools to Take You As Far As Your Vision®, The Way Ahead™, Powered by
Affymetrix™, GeneChip-compatible™, and Command Console™ are trademarks of Affymetrix, Inc.
All other trademarks are the sole property of their respective owners.
Limited Licenses
Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products, Affymetrix
grants you a non-exclusive, non-transferable, non-sublicensable license to use this Affymetrix product
only in accordance with the manual and written instructions provided by Affymetrix. You understand and
agree that except as expressly set forth in the Affymetrix terms and conditions, that no right or license to
any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this
Affymetrix product. In particular, no right or license is conveyed or implied to use this Affymetrix product
in combination with a product not provided, licensed or specifically recommended by Affymetrix for such
use.
Patents
Array Products may be covered by one or more of the following patents and/or sold under license from
Oxford Gene Technology: U.S. Patent Nos. 5,445,934; 5,700,637; 5,744,305; 5,945,334; 6,054,270;
6,140,044; 6,261,776; 6,291,183; 6,346,413; 6,399,365; 6,420,169; 6,551,817; 6,610,482; 6,733,977; and EP
619 321; 373 203 and other U.S. or foreign patents.
Reagent Products and their use may be covered by U.S. Patent Nos. 5,871,921, 5,866,337 and 6,858,412
and other U.S. or foreign patents that are owned or licensed by Affymetrix.
Copyright
©2008 Affymetrix, Inc. All rights reserved.
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Contents
CHAPTER 1
Getting Started. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
ABOUT YOUR DNA SAMPLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
General Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sources of Genomic DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
2
2
2
ABOUT THE PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Protocol Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
GENECHIP® SNP KITS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Contents and Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
TAQ POLYMERASE REQUIRED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Taq Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
PREVENTING SAMPLE CONTAMINATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Guidelines for Preventing Sample Contamination . . . . . . . . . . . . . . . . . . . . . . . . 7
ABOUT USING CONTROL DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About the Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Recommendations for Usage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Benefits of Using Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CHAPTER 2
8
8
8
8
Equipment, Supplies, Consumables. . . . . . . . . . . . . . . . . . .9
IMPORTANT NOTICE REGARDING PRODUCT SUPPORT . . . . . . . . . . . . . . . . . 10
About Qualifying for Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
ABOUT THE EQUIPMENT, SUPPLIES AND CONSUMABLES REQUIRED . . . . . 10
EQUIPMENT AND SOFTWARE SUPPLIED BY AFFYMETRIX FOR STANDARD
SYSTEM CONFIGURATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
EQUIPMENT, SUPPLIES, AND CONSUMABLES REQUIRED BUT
NOT PROVIDED . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vendor Contact Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment Required But Not Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Supplies Required But Not Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Consumables Required But Not Provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quality Control Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12
12
12
16
17
19
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CHAPTER 3
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Running the Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
BEFORE YOU START . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stages of the Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Normalize Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GeneChip® SNP Kits and Control DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Volumes For Mixes and Cocktails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pipets and Pipetting Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quality Control Gel Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
22
22
22
23
23
23
24
CREATE A PROJECT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prerequisites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary of Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About Sample Info Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About Sample Plate Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Create a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Create a New Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Add Sample Plates to a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
25
25
25
29
31
37
38
STAGE 1 – DESIGN AN ANNEAL PLATE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
How to Design an Anneal Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Viewing Anneal Plate Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
STAGE 2 – ANNEAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sample Concentration Requirement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kit Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thaw Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run an Anneal Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aliquot Samples from Sample to Anneal Plates . . . . . . . . . . . . . . . . . . . . . . . . .
Preparing Special Edition Assay Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare Anneal Cocktail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aliquot Anneal Cocktail and Begin Incubation . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare for Stage 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
43
43
44
44
44
45
45
46
48
49
50
51
52
STAGE 3 – PLAN AND RUN ASSAY PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Plan Assay Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run Assay Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
53
53
53
53
53
56
STAGE 4 – GAP FILL, DNTP, LIGATE, INVERT, FIRST PCR . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kit Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thaw The Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare The Gap Fill Mix, Exo Mix, and dNTP Plate . . . . . . . . . . . . . . . . . . . . .
57
57
60
60
61
61
61
61
contents
v
Add Gap Fill Mix – Addition #1 on Cold Block . . . . . . . . . . . . . . . . . . . . . . . . . .
Transfer and Split Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Add dNTPs – Addition #2 on Cold Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Add Exo Mix – Addition #3 on Cold Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Cleavage and Amp Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Add Cleavage Mix – Addition #4 on Thermal Cycler . . . . . . . . . . . . . . . . . . . . .
Add Amp Mix – Addition #5 on Thermal Cycler . . . . . . . . . . . . . . . . . . . . . . . . .
Transfer Assay Plates and Kit Components to Post-Amp Lab . . . . . . . . . . . . . .
62
63
65
66
67
68
69
69
STAGE 5 – SECOND PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kit Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Other Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thaw Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run Label Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare HY Allele Tube Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transfer Reactions to Label Plates and Add Allele Tube Mixes . . . . . . . . . . . . .
First Quality Control Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Viewing the First Quality Control Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
70
70
70
71
71
71
71
72
73
73
75
75
STAGE 6 – TARGET DIGEST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kit Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run Hyb Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Transfer Reactions to Hyb Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Add HY Digest Mix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Run the Meg HYDigest-A Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Second Quality Control Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Evaluating the Second Quality Control Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mark Experiments That Will Not Be Hybridized . . . . . . . . . . . . . . . . . . . . . . . . .
77
77
77
78
78
78
79
79
80
81
82
83
STAGE 7 – SAMPLE HYBRIDIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kit Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thaw Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the GeneChip® Universal Tag Arrays . . . . . . . . . . . . . . . . . . . . . . . . . .
Add the Hyb Cocktail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Denature the Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Load Reactions onto Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
84
84
84
84
85
85
86
87
87
88
STAGE 8 – STAIN AND WASH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kit Components Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prime the Fluidics Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Prepare the Storage Cocktail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Aliquot the Stain and Storage Cocktails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Load Arrays onto the Fluidics Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
92
92
92
92
92
93
94
94
94
95
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Remove Arrays from the Fluidics Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Process the Remaining Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Shut Down the Fluidics Station . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
STAGE 9 – SCAN ARRAYS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
About this Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Location and Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Equipment and Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Prepare the Scanner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Load the Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Scan the Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Adding Arrays During an AutoLoader Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Shutdown the Scanner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
CHAPTER 4
RESCANNING ARRAYS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Guidelines for Rescanning Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rewash Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Rescan the Array . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
103
103
103
103
DELETING PROJECTS AND PROJECT-RELATED INFORMATION . . . . . . . . . .
Deleting a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deleting Cluster Genotype Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deleting Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deleting Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deleting Anneal, Assay, Label and Hyb Plates . . . . . . . . . . . . . . . . . . . . . . . . .
Deleting Sample Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deleting Sample Info . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Deleting Assay Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
104
104
104
105
105
105
106
106
106
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107
GENECHIP® FLUIDICS STATION TROUBLESHOOTING . . . . . . . . . . . . . . . . . . 108
Fluid not Detected . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
TROUBLESHOOTING THE GENECHIP® SCANNER 3000 7G 4C . . . . . . . . . . .
Scanning Stopped Prematurely . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Autofocus Error Message . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Experiment not Hybridized Message . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111
111
112
112
TROUBLESHOOTING AND GENERATING MISSING .CEL FILES . . . . . . . . . . .
Causes of Missing .cel Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Determine that .cel Files are Missing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Locate .dat Filenames in GTGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Generate Missing .cel Files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
115
115
115
115
116
MANUALLY REGRIDDING ARRAYS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
What You Will See . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Locate the .dat File in GCOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Manually Aligning a Grid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Generate a .cel File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
118
118
119
120
121
contents
CHAPTER 5
vii
IDENTIFYING SKIPPED EXPERIMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
How To Identify Skipped Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Viewing the Import Experiment Data Window . . . . . . . . . . . . . . . . . . . . . . . .
Viewing the Experiment QC Summary Window . . . . . . . . . . . . . . . . . . . . . . .
122
122
122
123
TROUBLESHOOTING FAILED EXPERIMENTS . . . . . . . . . . . . . . . . . . . . . . . . . .
About Data Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Identifying Failed Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tools Available For Reviewing Your Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Troubleshooting Failed Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
124
124
124
125
126
Affymetrix GeneChip® Targeted Genotyping Analysis
Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .129
ABOUT AFFYMETRIX GENECHIP® TARGETED GENOTYPING
ANALYSIS SOFTWARE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
Software Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
SYSTEM MANAGEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
User Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Protocol Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Array Definition Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
130
130
135
138
PROJECT MANAGEMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
About Projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Array Definitions and Assay Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Delete Functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Export Functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
141
141
142
142
142
APPENDIX A
VENDOR CONTACT INFORMATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
APPENDIX B
THERMAL CYCLER PROGRAMS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pre-Amp Lab Thermal Cycler Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Post-Amp Lab Thermal Cycler Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Setting the Ramp Speed and Volume for Each Program . . . . . . . . . . . . . . . . .
148
148
148
148
MEG ANNEAL THERMAL CYCLER PROGRAM . . . . . . . . . . . . . . . . . . . . . . . . . 150
About the Meg Anneal Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
MEG 22 (20) CYCLE THERMAL CYCLER PROGRAMS . . . . . . . . . . . . . . . . . . . 152
About These Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
To Set Up the Meg 22 (20) Cycle Programs . . . . . . . . . . . . . . . . . . . . . . . . . . 153
MEG HYPCR THERMAL CYCLER PROGRAMS . . . . . . . . . . . . . . . . . . . . . . . . . 162
About the Meg Hypcr Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
MEG HYDIGEST-A THERMAL CYCLER PROGRAM . . . . . . . . . . . . . . . . . . . . . . 164
About the Meg HYdigest-a Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
MEG DENATURE THERMAL CYCLER PROGRAM . . . . . . . . . . . . . . . . . . . . . . 166
About the MEG Denature Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
viii
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
APPENDIX C
GUIDELINES FOR INCREASING SAMPLE THROUGHPUT . . . . . . . . . . . . . . . .
Equipment Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Resource Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Suggested Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
APPENDIX D
HOW TO CHANGE PLATE BARCODE RESTRICTIONS . . . . . . . . . . . . . . . . . . . 174
170
170
170
170
Chapter
1
Getting Started
2
About Your DNA Samples
SAMPLE CRITERIA
Genomic DNA Sample Criteria
• Concentration
All genomic DNA samples should be normalized to a single concentration of
150 ng/μL using 1X TE buffer. The Kit Control DNA included in every Affymetrix
GeneChip® SNP Kit has already been normalized to a working concentration.
• Minimum volume of DNA required
- For assay panels that are 5K and lower: 2 μg in approximately 15 μL of 1X TE
Buffer
- For assay panels greater than 5K: 4 μg in approximately 30 μL of 1X TE Buffer
• Buffer: 1X TE buffer
• Extraction and purification methods
- QIAGEN; QIAamp DNA Blood Maxi Kit
- Phenol-chloroform extraction
- Gentra PUREGENE
GENERAL REQUIREMENTS
Genomic DNA samples to be processed using GeneChip SNP Kits must:
• Not be amplified.
• Be free of inhibitors.
The extraction and purification method should render genomic DNA that is
generally salt-free. High concentrations of certain salts can inhibit enzyme reactions.
• Not be contaminated with other human genomic DNA sources or with genomic
DNA from other organisms.
• Not be highly degraded.
SOURCES OF GENOMIC DNA
The following sources of genomic DNA have been successfully processed with
GeneChip SNP Kits in our laboratories:
• Blood
• Cell line
• Semen
• Tissue including brain, liver and spleen
chapter 1 | Getting Started
3
About the Protocol
PROTOCOL OVERVIEW
One full run of the MIP Assay Protocol (not including data analysis) requires
approximately 2.5 days. The protocol is performed in two physically separated labs
referred to in this guide as the Pre-Amp Lab and the Post-Amp Lab. Separate rooms
are required to prevent contamination of sample DNA with amplified products from
previous reactions. To process samples at a throughput of 48 samples per day; 192
samples per 5 day week, at least two full-time employees (Research Assistants) are
required.
Recommendations for when to perform the various stages of the protocol are shown in
Figure 1.1 and Figure 1.2.
Figure 1.1
Recommended Workflow for Processing 48 Samples Per Day
4
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Figure 1.2
Recommended Timing for Stages of the Protocol
chapter 1 | Getting Started
5
GeneChip® SNP Kits
CONTENTS AND STORAGE CONDITIONS
GeneChip SNP Kits consist of the reagents and other components. One kit is
sufficient to process 24 reactions (including 1 control).
Kit components are shipped in multiple boxes. Because the various components must
be stored under different conditions, be sure to read each label to ensure that reagents
are properly stored.
Kit Components Used in the Pre-Amp Lab
The kit components listed in Figure 1.3 and Figure 1.4 are used in the Pre-Amp Lab
and should be stored at –20°C.
Assay Panel Box
1 tube each of the following:
• Assay Panel . . . . . . . 160 µL
• Kit Control DNA . . . . .70 µL
Note: This box will contain two assay
panel tubes if you are using a Special
Edition panel.
Figure 1.3
Assay Panel Box — Store at –20°C
Pre-Amp Box
1 tube each of the following:
• Enzyme A . . . . . . . . . . . . . . . . 60 µL
• Buffer A . . . . . . . . . . . . . . . . . 200 µL
• Water . . . . . . . . . . . . . . . . . . . . .2 mL
• Gap Fill Mix . . . . . . . . . . . . . . 130 µL
• Exo Mix . . . . . . . . . . . . . . . . . 530 µL
• Cleavage Tube . . . . . . . . . . . 3000 µL
• Cleavage Enzyme. . . . . . . . . . . .30 µL
• Amp Mix . . . . . . . . . . . . . . . . 3000 µL
• 1 plate dNTP Mix (20 µL per well)
Figure 1.4
Pre-Amp Box — Store at –20°C
6
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Kit Components Used in the Post-Amp Lab
The kit components listed in Figure 1.5, Figure 1.6 and Figure 1.7 are used in the
Post-Amp Lab. Post-Amp Box 1 kit components should be stored at –20°C.
Post-Amp Box 1
1 tube each of the following:
• HY A Allele Tube . . . . . . . . . . . 950 µL
• HY C Allele Tube . . . . . . . . . . . 950 µL
• HY G Allele Tube . . . . . . . . . . . 950 µL
• HY T Allele Tube . . . . . . . . . . . 950 µL
• HY Digest Mix . . . . . . . . . . . . . 260 µL
• Buffer H . . . . . . . . . . . . . . . . . . 330 µL
• Hyb Cocktail . . . . . . . . . . . . . . .900 µL
Figure 1.5
Post-Amp Box 1 — Store at –20°C
The Stain Cocktail (Figure 1.6) is shipped in a box labeled Stain Kit .
Even if the Stain Cocktail arrives frozen, it must be stored at 2–8°C.
Stain Kit Box
Stain Cocktail . . . . . 5 mL
Figure 1.6
Stain Kit Box — Store at 2–8°C
Wash Solution Kit contents (Figure 1.7) should be stored in the Post-Amp Lab at
room temperature.
Wash Solution Kit Contents
• Wash Solution A . . . . . . . . 500.0 mL
• Wash Solution B . . . . . . . . 500.0 mL
Figure 1.7
Wash Solution Kit — Store at room temperature
chapter 1 | Getting Started
7
Taq Polymerase Required
TAQ POLYMERASE
The MIP Assay Protocol has been optimized with and requires the use of following
two Taq polymerases:
Supplier
Description/Part Number
Amount/Vial
Clontech
TITANIUM™ / 639208
100 rxns
TITANIUM / 639209
500 rxns
TAQ Polymerase / 929197
200 µL
(100 µL sufficient to process 24 reactions)
Stratagene
(sufficient to process 48 reactions)
For Clontech and Stratagene contact information, refer to Appendix A, Vendor Contact
Information.
Preventing Sample Contamination
GUIDELINES FOR PREVENTING SAMPLE CONTAMINATION
To prevent sample contamination, we strongly suggest everyone entering the Pre- and
Post-Amp Labs follow these guidelines:
• Never bring any items that have been in the Post-Amp Lab into the Pre-Amp Lab.
Always assume that all surfaces in the Post-Amp Lab are contaminated with
amplified PCR product, and that all items that enter the Post-Amp Lab become
contaminated with product.
• Keep dedicated lab coats for the Pre-Amp Lab and the Post-Amp Lab.
• Use proper gowning procedures.
• Always wear gloves when handling equipment or samples in either lab.
• Do not move equipment between the Pre- and Post-Amp Labs. Keep dedicated
equipment in both labs.
• Print separate copies of the protocol for each lab.
• If both labs are connected by a door, do not return to the Pre-Amp Lab via the PostAmp Lab.
8
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
About Using Control DNA
ABOUT THE CONTROL DNA
The control DNA supplied in each GeneChip SNP Kit has been normalized to a
working concentration. No further dilution or concentration is required.
RECOMMENDATIONS FOR USAGE
To ensure the highest quality data, we strongly recommend running one positive
control for every 23 samples. Control DNA is included in every GeneChip SNP Kit.
Our protocol is written such that the control is always placed in column 12 of the PCR
plates (Anneal, Assay, Label and Hyb).
BENEFITS OF USING CONTROLS
Running control DNA is important for quality control and troubleshooting purposes.
Using controls can help you more quickly resolve issues related to:
• Sample quality
• Assay performance
Chapter
2
Equipment, Supplies, Consumables
10
Important Notice Regarding Product Support
ABOUT QUALIFYING FOR SUPPORT
The MIP Assay Protocol has been optimized for use on the Affymetrix GeneChip®
Scanner 3000 Targeted Genotyping System (GCS 3000 TG System) using the
equipment, supplies and consumables listed in this chapter.
A double asterisk (**) next to an item indicates that no substitutions can be made.
To qualify for support, these particular items must be used. In addition, training will
be conducted only when all of the equipment, supplies and consumables are
verified as present and properly installed.
About the Equipment, Supplies and Consumables Required
How the Information Is Organized
Information on the equipment, supplies and consumables required is organized as
follows:
• Equipment and Software Supplied by Affymetrix for Standard System Configuration
• Equipment, Supplies, and Consumables Required But Not Provided
- Equipment Required But Not Provided
- Supplies Required But Not Provided
- Consumables Required But Not Provided
- Quality Control Gels
chapter 2 | Equipment, Supplies, Consumables
11
Equipment and Software Supplied by Affymetrix for Standard
System Configuration
The following items are shipped to your facility directly from Affymetrix. A double
asterisk (**) next to an item indicates that no substitutions can be made.
Do not unpack any of the equipment supplied by Affymetrix. An Affymetrix
representative will unpack, install and verify the operation of the equipment and
software.
Store GeneChip® Universal Tag Arrays at 4°C.
Table 2.1
Equipment and Software Supplied by Affymetrix for Standard System Configuration
Item
** Affymetrix GeneChip® Scanner 3000 Targeted Genotyping
System including:
Quantity
Part Number
Laboratory
Location
1
00-0185
Pre and Post-Amp
Labs
As required
900602 (6 pk)
- GeneChip® Scanner 3000 7G 4C with 7G Instrument Control
Workstation
- GeneChip® Scanner 3000 Autoloader with 2D barcode reader
- GeneChip® Fluidics Station 450
- GeneChip® Hybridization Oven 640 with carriers
- GeneChip® Operating Software (GCOS), v1.4
- GeneChip® Targeted Genotyping Analysis Software, v1.5
- TG Pre-Amp Lab Workstation with 2D barcode reader
- TG Post-Amp Lab Workstation with SQL Server 2000 Standard
Edition license and 2D barcode reader
** The following GeneChip® Universal Tag Arrays as appropriate:
- GeneChip® Universal 3K Tag Array
Post-Amp Lab
900578 (96 pk)
- GeneChip® Universal 5K Tag Array
As required
900659 (6 pk)
Post-Amp Lab
900660 (96 pk)
- GeneChip® Universal 10K Tag Array
As required
900604 (6 pk)
Post-Amp Lab
900580 (96 pk)
- GeneChip® Universal 25K Tag Array
As required
900857 (6 pk)
Post-Amp Lab
900858 (96 pk)
12
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Equipment, Supplies, and Consumables Required But
Not Provided
VENDOR CONTACT INFORMATION
See Appendix A for vendor contact information.
EQUIPMENT REQUIRED BUT NOT PROVIDED
Table 2.2
Equipment Required but not Provided
Equipment
Quantity
Manufacturer/
Distributor
Part Number
(U.S.)
Laboratory
Location
** Thermal Cyclers – see Table 2.3 on page 13 for a list of thermal cyclers that have been validated for use with the MIP Assay
Protocol
Eppendorf ® Multipurpose Centrifuge 5804
2
VWR Intl.
53513-800
1 Pre-Amp Lab
1 Post-Amp Lab
Eppendorf ® Deepwell Swing Bucket Rotor with two
buckets
2
VWR Intl.
77888-214
1 Pre-Amp Lab
1 Post-Amp Lab
Mini Vortexer, analog
2
VWR Intl.
58816-121
1 Pre-Amp Lab
1 Post-Amp Lab
Galaxy Mini Centrifuge (interchangeable for
microtubes and strip tubes)
4
VWR Intl.
37000-700
2 Pre-Amp Lab
2 Post-Amp Lab
** Aluminum Block, 96-well
4
BioSmith
81001
Pre-Amp Lab
** Pipettes and pipette tips — see Table 2.4 on page 14 for a list of pipettes and tips that have been validated for use with the
MIP Assay Protocol
Carousel Stand for Rainin® Pipettes
4
Rainin
CR-7
2 Pre-Amp Lab
2 Post-Amp Lab
Portable Pipet-Aid (110V)
1
VWR
53498-103
Post-Amp Lab
Freezer, –20°C; deep freeze; manual defrost; 17 cu ft
2
—
—
1 Pre-Amp Lab
1 Post-Amp Lab
Refrigerator, 4°C refrigerator; 6 cu ft
2
—
—
1 Pre-Amp Lab
1 Post-Amp Lab
chapter 2 | Equipment, Supplies, Consumables
13
Thermal Cyclers
To run the MIP Assay Protocol at a throughput of 48 assays/day, you will need 4
thermal cyclers: 2 in the Pre-Amp Lab; 2 in the Post-Amp Lab.
The MIP Assay Protocol has been optimized using GeneAmp® PCR System 9700
Thermal Cyclers (96-well, gold-plated). The additional thermal cyclers listed in Table
2.3 have been validated for use with the protocol in the laboratory location specified.
Note the laboratory location for the thermal cyclers listed in Table 2.3. Some of
these instruments can be used in the Post-Amp Lab only.
Table 2.3
Thermal Cyclers Validated for Use With the MIP Assay Protocol
Manufacturer/
Distributor
Applied
Biosystems
Thermal Cyclers
BioRad
Thermal Cyclers
BioRad
Alpha Units
Item
Part Number
(U.S.)
GeneAmp® PCR System 9700 Thermal Cycler,
96-well, Silver
N8050001
GeneAmp® PCR System 9700 Thermal Cycler,
96-well, Gold-plated
4314878
GeneAmp® PCR System 9700 Thermal Cycler,
96-well, Aluminum
4314879
GeneAmp® PCR System 9700 Thermal Cycler,
Dual 96-well
4343176
DNA Engine® Peltier Thermal Cycler
chassis only; requires 1 Alpha Unit (see BioRad Alpha Units
below)
PTC-0200
DNA Engine Dyad® Peltier Thermal Cycler
chassis only; requires 2 Alpha Units (see BioRad Alpha Units
below)
PTC-0220
DNA Engine Dyad® Peltier Thermal Cycler
Disciple chassis only; requires 2 Alpha Units (see BioRad
Alpha Units below)
PTC-0221
DNA Engine Tetrad® 2 Peltier Thermal Cycler
Chassis only; requires 4 Alpha Units (see BioRad Alpha Units
below)
PTC-0240
Alpha™ Unit: Holds 96 x 0.2 ml tubes or one 96-well plate
ALS-1296
Moto Alpha™ Unit: Holds one 96-well plate with integrated
motorized heated lid
ALP-2296
Laboratory Location
Use in both the Preand Post-Amp Labs
Use in Post-Amp Lab
only
Use in Post-Amp Lab
only
14
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Pipettes and Pipet Tips
To run the MIP Assay Protocol, you will need the following types of pipettes. The
pipettes and tips validated fro use with the MIP Assay Protocol are listed in Table 2.4.
• Single channel pipettes
- 0.1–2 μL
- 0.5–10 μL
- 2–20 μL
- 20–200 μL
- 100–1000 μL
• 12-channel pipettes
- 0.5–10 μL or 1–20 μL
- 2–20 μL, 5–50 μL, 20–200 μL or 20–300 μL
• 24-channel pipettes, electronic or manual
- 2–20 μL
- 10–100 μL
Table 2.4
Pipettes and Pipet Tips Validated for Use With the MIP Assay Protocol
Manufacturer/
Distributor
Item
Quantity
Part Number
(U.S.)
Laboratory
Location
Pipet-Lite™ LTS™ Pipet, 0.1–2 µL
1
L-2
1 Pre-Amp Lab
Pipet-Lite LTS Pipet, 0.5–10 µL
2
L-10
1 Pre-Amp Lab
1 Post-Amp Lab
Pipet-Lite LTS Pipet, 2–20 µL
2
L-20
1 Pre-Amp Lab
1 Post-Amp Lab
Pipet-Lite LTS Pipet, 20–200 µL
2
L-200
1 Pre-Amp Lab
1 Post-Amp Lab
Pipet-Lite LTS Pipet, 100–1000 µL
2
L-1000
1 Pre-Amp Lab
1 Post-Amp Lab
LTS™ Pipette Tips, P-1000 barrier
(8 racks of 96 tips each; 768 tips per box)
As
required
RT-L1000F
(1 box)
LTS Pipette Tips, P-200 barrier
(10 racks of 96 tips each; 960 tips per box)
As
required
RT-L200F
(1 box)
LTS Pipette Tips, P-10 barrier
(10 racks of 96 tips each; 960 tips per box)
As
required
RT-L10F
(1 box)
SINGLE CHANNEL MANUAL PIPETTES
Rainin®
Rainin Pipet Tips
(use for single, 12-,
and 24-channel
Rainin pipettes)
Pre and Post-Amp
Labs
chapter 2 | Equipment, Supplies, Consumables
Manufacturer/
Distributor
Item
Quantity
Part Number
(U.S.)
Laboratory
Location
Pipet-Lite LTS 12 Channel Pipet, 0.5–10 µL
2
L12-10
1 Pre-Amp Lab
1 Post-Amp Lab
Pipet-Lite LTS 12 Channel Pipet, 2–20 µL
2
L12-20
1 Pre-Amp Lab
1 Post-Amp Lab
Pipet-Lite LTS 12 Channel Pipet, 20–200 µL
2
L12-200
1 Pre-Amp Lab
1 Post-Amp Lab
Pipetman® Ultra, U12-20, P20, 1–20 µL
2
F21041
1 Pre-Amp Lab
1 Post-Amp Lab
Pipetman Ultra, U12-300, 20–300 µL
2
F21043
1 Pre-Amp Lab
1 Post-Amp Lab
Gilson Diamond® sterilized extra long filter tips,
2-200 µL
As
required
D200ST
Gilson Diamond sterilized filter tips, 30–300 µL
As
required
DF300ST
Research®Series 2100, 0.5–10 µL
2
022453947
Research Series 2100, 10–100 µL
2
022453963
As
required
022491211
As
required
022491237
15
12-CHANNEL MANUAL PIPETTES
Rainin Pipettes
Gilson Pipettes
Pre- and Post-Amp
Labs
Gilson Pipet Tips
Eppendorf Pipettes
epT.I.P.S. Filter, 0.1–10 µL
Eppendorf Pipet
Tips
epT.I.P.S. Filter, 2–100 µL
1 Pre-Amp Lab
1 Post-Amp Lab
Pre- and Post-Amp
Labs
24-CHANNEL PIPETTES, MANUAL AND ELECTRONIC
Manual or electronic 24 channel pipettes can be used. Electronic recommended for better uniformity when mixing.
A minimum and maximum quantity of electronic pipettes is listed. If using the minimum, you will have to reprogram the
pipettes throughout the protocol. If using the maximum, you will not have to reprogram the electronic pipettes.
2–20 µL
- Pipet-Lite LTS 24 Channel Pipet (manual)
Rainin
- EDP3™ + Elect LTS™ Pipet 24 Channel
(electronic)
2
L24-20
2 to 4
E24-20
2
L24-100
2 to 4
E24-100
2 or 4
E3-RCS
1 Pre-Amp Lab
1 Post-Amp Lab
10–100 µL
- Pipet-Lite LTS 24 Channel Pipet (manual)
- EDP3+ Elect LTS Pipet 24 Channel (electronic)
E3 Rapid Charge Stand
1 Pre-Amp Lab
1 Post-Amp Lab
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
SUPPLIES REQUIRED BUT NOT PROVIDED
Table 2.5
Supplies Required but not Provided
Small Supplies
Quantity
Manufacturer/
Distributor
Part Number
Laboratory
Location
Cube Rack, polypropylene (4/pack)
2 packs
VWR Intl.
60985-444
1 Pre-Amp Lab
1 Post-Amp Lab
MicroTube Rack, 80-well (5/pack)
2 packs
VWR Intl.
10011-284
1 Pre-Amp Lab
1 Post-Amp Lab
96-place rack for 0.2 mL tubes (5/pack)
3 packs
USA Scientific
2300-9602
Pre- and PostAmp Labs
Preparation racks for PCR thin-walled tubes
(assorted colors)
1 case
Fischer Scientific
05-541-55
Pre- and PostAmp Labs
MiniAlarm Timer/Stopwatch
2
VWR Intl.
62344-585
1 Pre-Amp Lab
1 Post-Amp Lab
Lab Coats, dedicated
8
—
—
Pre and
Post-Amp Labs
Optional: Ice Bucket, red
1
VWR Intl.
35751-205
Post-Amp Lab
1
VWR Intl.
35751-216
Post-Amp Lab
1
VWR Intl.
35751-218
Pre-Amp Lab
2
—
—
1 Pre-Amp Lab
1 Post-Amp Lab
2 pairs
—
—
1 Pre-Amp Lab
1 Post-Amp Lab
—
—
—
Post-Amp Lab
Note: You will need an ice bucket dedicated to the
Post-Amp Lab.
Optional: Ice Container, red (8.5” x 16” x 3”)
Note: You will need an ice container dedicated to the
Post-Amp Lab.
Optional: Ice Container, green (8.5” x 16” x 3”)
Note: You will need an ice container dedicated to the
Pre-Amp Lab.
Calculator
Scissors
Distilled, deionized water supply or carboys (for
cleaning the fluidics station)
chapter 2 | Equipment, Supplies, Consumables
17
CONSUMABLES REQUIRED BUT NOT PROVIDED
Affymetrix GeneChip® SNP Kits
One GeneChip SNP Kit includes reagents sufficient to process 24 reactions. The
specific kit required to process your samples is determined by the assay panel you are
using.
Kit components are shipped in multiple boxes. Store each box as directed on the
label.
Additional Consumables
The quantities listed for consumables are sufficient to process 600 reactions including
controls.
Table 2.6
Additional Consumables – Quantities sufficient to process approximately 600 reactions
Item
Quantity
Manufacturer
/Distributor
Part Number
(U.S.)
Laboratory
Location
- Stratagene Taq DNA Polymerase, 200 µL/vial
As needed
Stratagene
929197
(1 vial)
Pre-Amp Lab
- Clontech TITANIUM™ Taq Polymerase
As needed
Clontech
639208
(100 rxns)
Post-Amp Lab
** Both of the Taq DNA Polymerases listed below are
required.
100 rxn size is sufficient for processing 24 reactions
(with 10% extra)
639209
(500 rxns)
** PCR Plates (96-well) and barcode labels — see PCR Plates on page 18 and Table 2.7 on page 19 for the PCR plate vendors
and barcode label supplier validated for use with the MIP Assay Protocol.
** Clear Film for Arrays (81 labels/sheet; 10 sheets per pack)
1 pack
Affymetrix
Post-Amp Lab
** MicroAmp® Clear Adhesive Films (100 films/pk)
3 pks
Applied
Biosystems
4306311
(1 pack)
Pre and PostAmp Labs
Pipettes, 5 mL, disposable, individually wrapped (200/case)
1 case
VWR
20171-046
Post-Amp Lab
Pipettes, 10 mL, disposable, individually wrapped (200/case)
1 case
VWR
20171-042
Post-Amp Lab
PCR 12-well Tube Strips with Strip Bubble Caps (80/pack)
1 pk
VWR
53509-306
(1 pack)
Pre and PostAmp Labs
Eppendorf Color-Coded Safe-Lock Microcentrifuge Tubes,
amber, 2.0 mL (500/case)
2 cases
VWR
20901-541
Post-Amp Lab
Eppendorf Color-Coded Safe-Lock Microcentrifuge Tubes,
natural, 2.0 mL (500/case)
2 cases
VWR
20901-540
Pre and PostAmp Labs
Reagent Reservoirs, 50 mL (200/case)
1 case
VWR Intl.
53504-035
(1 case)
Pre and PostAmp Labs
Microtube Tough Spots® for 0.5–2.0 mL tubes, white
(1000/roll)
1 roll
USA Scientific
9185-0000
(1 roll)
Post-Amp Lab
18
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Table 2.6
Additional Consumables – Quantities sufficient to process approximately 600 reactions
Item
Quantity
Manufacturer
/Distributor
Part Number
(U.S.)
Laboratory
Location
1 case
VWR Intl.
21008-670
(1 case)
Pre and PostAmp Labs
6 boxes
—
—
Pre and PostAmp Labs
Bench Pads
1 case
VWR Intl.
56616-026
Pre and PostAmp Labs
Kimwipes® (12” x 12”)
2 packs
VWR Intl.
21905-011
(1 pack)
Pre and PostAmp Labs
Marking pens, one of the following types
• Water soluble
• Fluorescent
1 pack
Fischer
Scientific
13-380-15A
or
13-384
Post-Amp Lab
12 pens
—
—
6 Pre-Amp Lab
6 Post-Amp
Lab
1 pack
—
—
Post-Amp Lab
1 roll
—
—
Post-Amp Lab
Compressed air (canned)
2 cans
—
—
Post-Amp Lab
Tacky Floor Mats
1 case
VWR Intl.
12777-112
(1 case)
Post-Amp Lab
Corning Conical Tubes, 15 mL (500/case)
Gloves, 100/box
Marking pens, permanent, fine point, assorted colors
Razor blades
Aluminum foil
PCR Plates
To run the MIP Assay Protocol, you will need a supply of 96-well PCR plates with
specific barcode labels. To facilitate sample tracking, the plates are referred to using
specific terminology throughout the various stages of the protocol. These designations
and the barcode prefix associated with each type of plate is listed below.
• Anneal Plate – ANN barcode
• Assay Plates – ASY barcode
• Label Plates – LBL barcode
• Hyb Plates – HYB barcode
If you purchase plates from a vendor other than Axygen, you must purchase
barcode labels from Axygen to apply to your plates. The plates supplied by Axygen
come with the barcode labels already affixed to them.
chapter 2 | Equipment, Supplies, Consumables
19
Table 2.7
PCR Plates and Barcode Labels Validated for Use With the MIP Assay Protocol – Quantities sufficient to process approximately
600 reactions
Manufacturer/
Distributor
Item
Quantity
Part Number
(U.S.)
Laboratory
Location
Anneal PCR Plates, 96-well thin-wall
(with ANN barcode affixed; 50/case)
1 case
321-63-ANN
(1 case)
Pre-Amp Lab
Assay PCR Plates, 96-well thin-wall
(with ASY barcode affixed; 50/case)
1 case
321-63-ASY
(1 case)
Pre-Amp Lab
Label PCR Plates, 96-well thin-wall
(with LBL barcode affixed; 50/case)
1 case
321-63-LBL
(1 case)
Post-Amp Lab
Hybridization PCR Plates, 96-well thin-wall
(with HYB barcode affixed; 50/case)
1 case
321-63-HYB
(1 case)
Post-Amp Lab
PCR Plate, 96 Well, Half Skirt
(no barcode; 40/case)
8 cases
EK-19280
Pre- and PostAmp Labs
BC-LABEL-SMP
Pre-Amp Lab
BC-LABEL-ANN
Pre-Amp Lab
BC-LABEL-ASY
Pre-Amp Lab
Barcode Labels for Label Plates
BC-LABEL-LBL
Post-Amp Lab
Barcode Labels for Hyb Plates
BC-LABEL-HYB
Post-Amp Lab
PCR PLATES
Axygen
distributor
Greiner
(from E & K
Scientific)
Note: If purchasing Greiner plates, you must also
purchase barcode labels from Axygen (listed
below)
BARCODE LABELS
Axygen
distributor
Barcode Labels for Sample Plates
Barcode Labels for Anneal Plates
Barcode Labels for Assay Plates
As
required
QUALITY CONTROL GELS
Training Requirements
As part of the training session, quality control (QC) gels are run on each plate of
samples processed. Running QC gels helps the trainer determine the source of issues
that may arise.
Your laboratory must have gel electrophoresis capability and a gel imaging system.
Gel recommendations are listed below.
QC Gel Recommendations
We recommend running two QC gels per assay:
• While learning to run the MIP Assay Protocol
• For troubleshooting purposes
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Precast Gel Recommendations
We recommend using one of the following precast gels and gel system:
Biorad Precast Gels and Gel Box System
Table 2.8
Recommended Biorad Precast Gels and Gel Box System
Item
Part Number
For Training
Ongoing
Wide Mini ReadySub-Cell GT Cell
170-4489
Two cells required to
concurrently run two gels
Two cells required to
concurrently run two gels
ReadyAgarose 96 Plus 3% TBE Gel
161-3062
8 gels
1 gel/24 assays
Invitrogen Precast Gels and Gel System
Table 2.9
Recommended Invitrogen Precast Gels and Gel System
Item
Power supply and gel tray
recommendations:
• One Mother Base
• Three Daughter Bases
E-Gel 48 4% Agarose (8 gels/box)
Part Number
For Training
Ongoing
Mother Base: EB-M03
Daughter Bases: EB-D03
One Mother Base and
Three Daughter Bases
One Mother Base and
Three Daughter Bases
G8008-04
8 boxes
2 gels/24 assays
Self-Cast Gel Recommendations
If casting your own gels, we recommend following these guidelines:
• Use acrylamide or a high-resolution agarose.
• For agarose gels, we recommend preparing a 4% Agarose SFR gel using
Agarose SFR by Amresco, part number J234-100G.
Chapter
3
Running the Protocol
22
Before You Start
STAGES OF THE PROTOCOL
The MIP Assay Protocol is presented and performed in stages. These stages are:
• Stage 1 – Design an Anneal Plate
• Stage 2 – Anneal
• Stage 3 – Plan and Make Assay Plates
• Stages 4 – Gap Fill, dNTP, Ligate, Invert, First PCR
• Stage 5 – Second PCR
• Stage 6 – Target Digest
• Stage 7 – Sample Hybridization
• Stage 8 – Stain and Wash
• Stage 9 – Scan Arrays
To prevent sample contamination from PCR products, stages 1, 2, 3 and 4 are
performed in the Pre-Amp Lab. The remaining stages are performed in the Post-Amp
Lab.
NORMALIZE SAMPLES
All genomic DNA samples should be normalized to a single concentration of
150 ng/μL using 1X TE buffer. The Kit Control DNA included in every
GeneChip® SNP Kit is already normalized to a working concentration.
Minimum volume of DNA required:
• For assay panels that are 5K and lower: 2 μg in approximately 15 μL of 1X TE Buffer
• For assay panels greater than 5K: 4 μg in approximately 30 μL of 1X TE Buffer
For more information on sample criteria and recommendations, see Sample Criteria on
page 2.
GENECHIP® SNP KITS AND CONTROL DNA
One GeneChip SNP Kit is sufficient to process 24 reactions (23 samples and 1
control).
To ensure the highest quality data, we strongly recommend running one positive
control for every 23 samples. Control DNA is included in each GeneChip SNP Kit.
chapter 3 | Running the Protocol
23
ENZYMES
The enzymes in GeneChip SNP Kits are temperature sensitive. They must be kept at
−20°C until used.
For all mixes and cocktails, always add the enzymes last, and add them right before
you plan to use the mix or cocktail.
VOLUMES FOR MIXES AND COCKTAILS
The volumes specified for each mix or cocktail are given for one of the following:
• 24 or 48 reactions
• 24 or 48 arrays
PIPETS AND PIPETTING RECOMMENDATIONS
The types of pipettes used throughout this protocol are:
• Single channel, manual
• 12-channel, manual
• 24-channel, manual or electronic
When the protocol calls for the use of 24-channel pipettes, you can use either a manual
or an electronic pipette. Rainin EDP3-Plus with LTS electronic pipettes are
recommended for better uniformity when mixing.
General Pipetting Recommendations
To ensure full volume transfer, check pipette tips after each pickup and dispense. To
avoid the formation of air bubbles, dispense liquids at the bottom of each well. Always
use the type and volume of pipette specified in the protocol.
Electronic Pipetting Recommendations
Follow the instructions provided with the pipettes for the dispense/mix program that:
• Allows reagent to be aspirated and dispensed at a set volume
• Mixes automatically upon dispensing wherein the mix volume can be different from
the dispense volume
Two options are available for tracking the number of mixes when using Rainin EDP3Plus electronic pipettes: the counter option, or the beep option (pipette beeps after
each mix). We recommend using the beep option, since the counter does not start at
zero with each use. Instead, it counts pipette operations sequentially. Refer to the
instructions provided with the pipettes for more information.
24
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
QUALITY CONTROL GEL RECOMMENDATIONS
We recommend running quality control gels at two points during the protocol.
Knowing in advance that a sample will not provide data will save arrays. The purpose
of each gel is described below.
• Gel 1: Run to identify any samples that did not amplify.
No bands are visible for samples that have not amplified.
• Gel 2: Run to confirm that bands have digested after the target digest stage.
For more information on quality control gels, see:
• First Quality Control Gel on page 75
• Viewing the First Quality Control Gel on page 75
• Second Quality Control Gel on page 81
• Evaluating the Second Quality Control Gel on page 82
chapter 3 | Running the Protocol
25
Create a Project
PREREQUISITES
Before you can create a project and run the protocol, you must import the following
into the Affymetrix GeneChip® Targeted Genotyping Analysis Software (GTGS).
• Array definitions
Array definitions are loaded into the GTGS during system installation. They are also
stored on the CD-ROM included with the software. See Array Definition Management
on page 138 for more information.
• Protocol definitions
Protocol barcodes are supplied on the MIP Assay Protocol Barcodes quick reference
card. See Adding Protocols on page 135 for more information.
SUMMARY OF STEPS
To create a project, you will:
Optional: Create a Sample Info file that contains sample information such as
species and gender. If gender is important to your study, consider creating Sample
Info files. See About Sample Info Files on page 25.
2. Create at least one Sample Plate file containing sample plate layout information
(one file per sample plate)
3. Create a new project in GTGS by importing:
- Assay panel file – located on the CD-ROM received with your first shipment of
GeneChip SNP Kits for a particular assay panel.
- Genotype settings file – located on the same CD-ROM as the assay panel file
1.
- Sample info files
- Sample plate files
4. Add sample plates to your project
5.
Design an anneal plate
ABOUT SAMPLE INFO FILES
Creating a Sample Info file is optional. These files can contain the following
information:
• Sample name (mandatory)
• Sample gender (optional)
• Population (optional)
• Species (optional)
If your assay panel can report gender, and if sample gender is important, consider
creating Sample Info files that include the gender of each sample. This information can
help troubleshoot sample tracking errors by comparing the known gender with the
26
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
inferred gender. The inferred gender is called by the software during data analysis. Refer
to the Affymetrix GeneChip® Targeted Genotyping Analysis Software User Guide for more
information.
Once a sample info file has been imported, you can add to or update the existing
information. You can also delete sample information that ends up not being used.
Creating Sample Info Files
Sample information must be entered into a tab-delimited text file using an application
such as Microsoft® Office Excel® (saved as a .txt file) or Microsoft® Notepad.
If you decide not to create Sample Info files, the sample name only will be appear
automatically in the Sample Info table. Gender, population and species will be
designated as Unknown (Figure 3.1).
Figure 3.1
Sample Info Icon and Table
Guidelines to follow when creating Sample Info files are listed below.
• Use the column headings and format shown in Figure 3.2.
• Samples names:
- Maximum length is 50 characters
- Can appear only once in a Sample Info file
- Cannot contain the following characters: \ / : ; * ? " < > | ' , { } [ ]
chapter 3 | Running the Protocol
27
Figure 3.2
Example of Sample Info Files
Adding To or Updating Sample Info Files
You can add to and update information that has already been imported from Sample
Info Files.
To add to and update Sample Info File information:
1.
2.
3.
4.
5.
6.
7.
8.
Edit the sample info text file.
In the left pane of GTGS, open the menu and select Samples.
Right-click the Sample Info icon and select Add/Update Sample Info.
In the Add/Update Sample Info window, click the Browse button.
Locate and select the Sample Info filename.
Select one or both check boxes to Add New Sample Info or Update Existing
Sample Info.
Click Import.
Click Save.
28
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Figure 3.3
Add/Update Sample Info Window
Removing Unused Sample Info
You can remove information that was imported from Sample Info Files but was not
used in your study.
To remove unused sample info:
In the left pane of GTGS, open the menu and select Samples.
2. Right-click the Sample Info icon and select Remove Unused Sample Info.
3. Click Yes when prompted to confirm this action.
1.
chapter 3 | Running the Protocol
29
ABOUT SAMPLE PLATE FILES
The layout of each sample plate used in the MIP Assay Protocol must be imported into
GTGS prior to sample processing. The information in these files is used to design
Anneal Plate layouts for the first stage of the protocol.
Sample plate information can be updated until the barcode of an Anneal Plate
containing a sample from a particular plate is scanned.
Creating Sample Plate Files
The layout of each sample plate must be entered into a tab-delimited text file using
an application such as Microsoft Office Excel (saved as a .txt file) or Notepad.
• Use the column headings and format shown in Figure 3.4 on page 30. Sample types
can be sample, control and water.
• Each text file should correspond to one sample plate only.
• Well designations must be based on a 96-well plate format (A1 through H12.)
• The sample plate does not need to be full. Do not include empty wells in the file.
• Samples name guidelines:
- Must be unique for each unique DNA (repeats should have the same name)
- Maximum length is 50 characters
- Cannot contain the following characters: \ / : ; * ? " < > | ' , { } [ ]
Assign a unique name to each unique DNA. Repeated samples of the same DNA
should have the same name. GTGS compares samples based on sample name. If
different DNA samples are assigned the same name, and if those samples are
analyzed together, the repeatability metrics calculated by GTGS will be incorrect.
For text files containing control DNA information, we recommend including the lot
number in the filename. For example, SMP2_wo0260_KitControl.txt. You can use
this filename as a the sample plate barcode later in the protocol.
Updating Sample Plate Information
Sample plate information, except for the sample name, can be updated at any time.
To update sample plate information:
Edit the sample plate text file.
2. In the left pane of GTGS, open the menu and select Samples.
3. Expand the Sample Plates icon.
1.
4.
5.
6.
7.
8.
Right-click a sample plate and select Update Sample Plate …
In the Update Sample Plate window, click the Browse button.
Locate and select the sample plate filename.
Select External or Internal Samples as appropriate.
Click Import.
30
9.
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Click Save.
Control DNA Recommendations
To ensure the highest quality data, we strongly recommend running one positive
control for every 23 samples. Control DNA is included in each GeneChip SNP Kit.
Microsoft Excel files with
sample plate layout and
control information.
These files must be saved
as tab-delimited text files
(.txt).
Figure 3.4
Example of Text Files with Sample Plate Layout and Control DNA Information
chapter 3 | Running the Protocol
31
CREATE A PROJECT
Import Assay Panel and Genotype Settings Files
You must import the file created for your assay panel before you can run the protocol.
The file is located on the CD-ROM that was shipped with the first GeneChip SNP
Kits you received.
At the same time, we strongly recommend importing the genotype settings file that
is located on the same CD-ROM. Even though the information in this file will not be
used until data analysis, the software will not prompt you to load it. The settings in
this file were optimized for your particular assay panel. If it is not present when
performing data analysis, default genotype settings will be used and your results may
not be optimal.
To import assay panel and genotype settings files:
1.
In GTGS, import the assay panel and genotype settings files as follows:
Load the CD-ROM containing the files into the hard drive.
B. Open the drop-down menu and select Assay Panels.
A.
Figure 3.5
Selecting Assay Panels from the Menu
C.
D.
E.
F.
G.
H.
Open File → Import Assay Panel (or right-click the Assay Panels folder)
In the Import Assay Panel window, click the Browse button next to the Assay
Panel File field.
Locate and select the assay panel filename, then click Open.
Click the Browse button next to the Genotyping Settings File field.
Locate and select the genotype settings filename, then click Open.
Click the Import button.
Notice that the table in the Import Assay Panel window is now populated
(Figure 3.6).
32
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Figure 3.6
Importing the Assay Panel and Genotype Settings Files
Optional: Enter a description in the Description field.
J. Click Save.
If you expand the Assay Panel icon in the left pane, the type of GeneChip® Universal
Tag Array that must be used with your assay panel is displayed. In the right pane, a
list of the features present on the array is displayed.
I.
Figure 3.7
Displaying the Type of Array to be Used with a Particular Assay Panel
chapter 3 | Running the Protocol
33
Import Sample Information
You will now import your sample info files (optional) and sample plate files (required).
To import sample information:
Open the drop-down menu and select Samples.
2. Optional: Import Sample Info files as follows:
1.
A.
Right-click the Sample Info icon and select Add/Update Sample Info.
Figure 3.8
Importing Sample Info Files
B.
C.
D.
E.
F.
G.
In the Add/Update Sample Info window, click the Browse button next to the
Sample Info File field (Figure 3.9).
Locate and select the Sample Info filename, then click Open.
Select the Add New Sample Info check box.
Click the Import button.
Notice that the Sample Info Preview table is now populated.
Repeat this procedure for all Sample Info files you want to import.
Click Save.
Figure 3.9
Add/Update Sample Info Window
34
3.
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Import Sample Plate files as follows:
A.
Right-click the Sample Plates icon and select Import Sample Plate (Figure
3.10).
Figure 3.10
Importing Sample Plates
In the Import Sample Plate window (Figure 3.11), click the Browse button.
C. Locate and select a sample plate filename, then click Open.
D. Select Internal or External Samples as appropriate.
E. Click Import.
Notice that information about the sample plate is now displayed in the Import
Sample Plate table (Figure 3.11). The software automatically designates the
sample plate filename as the barcode.
B.
Figure 3.11
Import Sample Plate Window
To change the barcode, either scan the sample plate barcode or type a new
barcode into the Barcode field.
G. Click Save.
4. Repeat this procedure for each sample plate you want to add.
F.
chapter 3 | Running the Protocol
35
About Sample Plate Information
Expand the Sample Plates folder in the left pane to see a list of the sample plates you
have imported. Select a sample plate name to see information specific to a particular
plate.
In the right pane, you can view:
• General information about each sample plate such as the date it was imported
(Sample Plates tab; Figure 3.12).
• A list of samples on a particular plate by selecting the plate name, then clicking the
Sample Plate Wells tab (Figure 3.13).
• The Sample Info associated with a particular sample plate by selecting the name of
the sample plate and clicking the Sample Info tab (Figure 3.14).
You can fail a sample plate and add a description for a sample plate by modifying the
sample plate properties. See Modifying Sample Plate Properties on page 36.
Figure 3.12
Sample Plate Information
.
Figure 3.13
Sample Plate Wells Tab
36
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Figure 3.14
Sample Info Tab
Modifying Sample Plate Properties
You can add a description for a sample plate or fail the entire sample plate by
modifying the sample plate properties.
To modify sample plate properties:
Select a sample plate, then open Edit → Properties (or right-click the sample
plate name).
2. Do one of the following:
1.
Add or modify a description in the Description field (Figure 3.15).
B. Select or deselect the Failed check box.
3. Click Save.
A.
Figure 3.15
Modifying Sample Plate Properties
chapter 3 | Running the Protocol
37
CREATE A NEW PROJECT
Open the drop-down menu and select Projects.
2. Right-click the Projects folder and select New Assay Project.
1.
3.
In the Select an Assay Panel window (Figure 3.16), select the name of an assay
panel, then click OK.
Figure 3.16
Select an Assay Panel Window
4.
In the Project window (Figure 3.17):
A.
Enter a project name in the Name field.
Project name constraints are:
- Maximum length is 50 characters
- Cannot contain the following characters: \ / : ; * ? “ < > | ' , {} [ ]
Projects are displayed alphabetically by name. Keep this in mind if you want your
projects displayed in a particular order.
B.
Accept the default values for Scanner Type (GeneChip_Scanner_3000) and
Color Format (Four_Color).
C.
Optional: In the Description field, enter a description for the project.
Figure 3.17
Project Window
D.
Click Save.
38
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
In the left pane, click the + icons next to the project and Tracking icons to expand
them (Figure 3.18). Notice the placeholders that have been created under the Tracking
icon (Sample Plates, Anneal Plates, Assay Plates, Label Plates, Hyb Plates, and
Arrays). Placeholders for array data information and genotype results are also created.
Figure 3.18
Structure of a Project
ADD SAMPLE PLATES TO A PROJECT
To add sample plates to a project, you will:
• Import a Sample Plate file (see About Sample Info Files on page 25 for more
information)
• Scan the barcode of the sample plate
• Add the sample plate to your project
Add Sample Plates to Your Project
To add sample plates to your project:
Expand your project icon, then expand the Tracking icon (Figure 3.19).
2. Right-click the Sample Plates folder, and select Add Existing Sample Plate.
1.
chapter 3 | Running the Protocol
Project icon
Tracking icon
Figure 3.19
Adding Sample Plates to a Project
Select a sample plate, then click OK (Figure 3.20).
4. Repeat this procedure to add more sample plates to your project.
3.
Figure 3.20
Selecting a Sample Plate
To view the sample plates added to your project, expand the Sample Plates icon.
Sample plate information is also displayed in the right pane of the window.
39
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Stage 1 – Design an Anneal Plate
Information describing the samples and their well locations on each sample plate is
used to design the Anneal Plate. This information is imported into GTGS from tabdelimited text files. If the sample information you require is not yet in GTGS, import
it now. Refer to the appropriate sections under Create a Project starting on page 25 to
create sample files and import them into your project.
For Stage 1, sample plate information is used to design an Anneal Plate. You will
designate:
• Which samples are to be transferred from a sample plate to an Anneal Plate.
• Where the samples are to be located on the Anneal Plate.
HOW TO DESIGN AN ANNEAL PLATE
To design an Anneal Plate:
Right-click the Anneal Plates icon for your project and select Add Anneal Plate.
2. In the Define Sample Plate to Anneal Plate Mapping window, open the Source
menu and select a sample plate (Figure 3.21).
1.
Figure 3.21
Selecting a Sample Plate for Anneal Plate Design
3.
Specify which samples are to be transferred from the Sample Plate to the Anneal
Plate as follows:
A.
Click and drag your cursor over one or more wells on the Sample Plate.
Click and drag your cursor over the Anneal Plate wells that you want the
samples loaded in. (Shown in Figure 3.23 on page 41.)
C. Click Set.
Notice that the transfers you designate are displayed at the bottom of the
window in the Transfers table (Figure 3.22).
4. When finished, click Save.
5. To edit the layout after you have clicked Save:
B.
A.
B.
C.
D.
E.
Expand the Anneal Plates icon.
Right-click the name of the Anneal Plate and select Edit Sample Mapping.
Select a line in the Transfers table; then click Clear (Figure 3.22).
Make your changes.
Click Save.
chapter 3 | Running the Protocol
41
Transfers with the status Ready can be changed. Transfers with the status
Complete cannot be changed.
To modify transfers, select the appropriate line in the Transfers table and click Clear.
Transfers with the status Ready can be changed. Transfers with the status Complete cannot be changed
Figure 3.22
Making Changes to Sample Mapping
In this example, DNA from sample plate SMP2_0000601, well A1 to A11, B1 to B12, C1 to C11, and D1 to D12 is
to be loaded into Anneal Plate wells A1 to A11, B1 to B12, C1 to C11 and D1 to D12. Kit Control DNA from the
tube named SMP2_wo0260_KitControl is to be loaded into Anneal Plate wells A12 and C12.
Figure 3.23
Example of Sample to Anneal Plate Mapping
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
VIEWING ANNEAL PLATE INFORMATION
To view the Anneal Plates you have designed, expand the Anneal Plates icon. To view
information about a particular Anneal Plate, select the Anneal Plate name. In the right
pane, select the appropriate tab to view:
• Properties – lists properties for an Anneal Plate such as who created it, the date it
was created and the barcode.
• Incoming transfers – indicates which samples are to be loaded onto the Anneal Plate
from a particular sample plate. Information includes the sample plate well and the
Anneal Plate well.
• Outgoing transfers – indicates which Anneal Plate reactions are to be transferred to
a specific Assay Plate and where they are to be placed (well location) on the Assay
Plate. This table will remain empty until you have run the Anneal Plate and created
an Assay Plate.
• All experiments – provides information that is available after the array barcodes are
scanned. This information includes the date each sample was hybridized onto an
array, as well as the barcode and well for each plate the sample was on during the
protocol.
chapter 3 | Running the Protocol
43
Stage 2 – Anneal
ABOUT THIS STAGE
During this stage, the assay panel probes are annealed to genomic DNA target
samples.
DNA Cleavage
Site 2
DNA Cleavage
Site 1
Genomic
Homology 1
PCR Primer
Sites
Tag
Hybridizes to
the array
Genomic
Homology 2
Figure 3.24
Assay Panel Probe
Genomic DNA samples, the assay panel probes, and reagents (Anneal Cocktail) are
mixed in an Anneal Plate. An Anneal Plate is a 96-well PCR plate with the barcode
designation ANN<barcode>. The plate is then placed on a thermal cycler and the
program, Meg Anneal, is run. Because the samples must be left to anneal for ≥ 16 hours,
this stage is typically started at the end of the day, and the anneal program is allowed
to run overnight.
SNP of
interest on
genomic DNA
Strand 1
Tag sequence that hybridizes to
GeneChip® Universal Tag Array
Cleavage site #1
PCR primers
Cleavage site #2
3'
5' 3'
5'
3'
5'
Complimentary genomic DNA homology regions 1 & 2
Figure 3.25
Assay Panel Probe Annealed to Genomic DNA Sample
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
LOCATION AND DURATION
• Pre-Amp Lab
• Hands-on time: approximately 45 minutes
SAMPLE CONCENTRATION REQUIREMENT
Before starting this procedure, all samples should be normalized to a single
concentration of 150 ng/μL using 1X TE buffer.
The Kit Control DNA included in every GeneChip SNP Kit has already been
normalized to a working concentration. No further dilution or concentration is
required. For more information, see Normalize Samples on page 22.
EQUIPMENT AND MATERIALS REQUIRED
The following equipment and materials are required to perform this stage. Quantities
shown are for processing 48 reactions.
Table 3.1
Equipment and Materials Required for Stage 2 - Anneal
Quantity
Item
1
Centrifuge, plate
1
Cube rack
1
Eppendorf tube, 2 mL
1
Ice container, rectangular, filled with ice
1
Marking pen, extra fine point, permanent
3
MicroAmp Clear Adhesive Films
1
Microfuge
1
PCR plate, 96-well, with ANN barcode
2–3
Plate holders (preparation racks)
1
Reagent reservoir, 50 mL
48
Samples of genomic DNA in barcoded plates
1
Thermal cycler, 96-well GeneAmp PCR System 9700
1
Vortexer
chapter 3 | Running the Protocol
45
KIT COMPONENTS REQUIRED
Two GeneChip SNP Kits are required to anneal 48 reactions (including controls). The
following components are required from each kit:
• Water (P/N 5-0004)
• Assay Panel
For Special Edition panels, there will be two assay panel tubes in the kit:
- Standard Assay Panel tube (fill size 160 μL)
- Supplemental Assay Panel tube (fill size 60 μL)
• Buffer A
• Enzyme A
• Kit Control DNA
THAW REAGENTS
To thaw reagents:
Place the Water from kit, all Assay Panel tubes, Buffer A, and Kit Control DNA
in a cube rack.
2. Allow to warm to room temperature on the bench top.
3. Keep on ice until ready to use.
1.
Leave Enzyme A at –20°C until ready to use.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
RUN AN ANNEAL PLATE
To run an Anneal Plate:
If a project has not been created, create one now.
Creating a project is typically done once at the beginning of a study. You must
create at least one project before you can perform the protocol and track samples
using GTGS. For instructions, see Create a Project on page 25.
2. Using a permanent marking pen, label the ANN-barcoded PCR plate as shown in
Figure 3.26.
Four full rows (48 reactions) from an Anneal Plate are required to fill two Assay
Plates.
1.
SAMPLES
C X
X
C X
X
C = Control DNA. Use of
control DNA is optional.
IMPORTANT To ensure the
highest quality data, we
strongly recommend running
1 control for every 23
samples.
Placement of samples
shown in wells 1 through 11
of rows A, and C, and wells 1
through 12 of rows B and D.
X is a placeholder. Mark the Anneal Plate with a sample
plate row designation that is meaningful to you.
Figure 3.26
Anneal Plate Labeling and Sample Placement
In GTGS, expand the Project, Tracking and Anneal Plates icons.
4. Do the following:
3.
A.
Right-click an Anneal Plate and select Run Anneal Plate.
If you are not sure which Anneal Plate to choose, select an Anneal Plate in the
left pane. In the right pane, click the Incoming Transfers tab and view the
information displayed.
In the Run Anneal Plate window, follow the prompts in the uppermost field
and scan the barcode of the first sample plate listed (Figure 3.27).
C. Press Enter or click Next.
D. If more than one sample plate is being used, scan the remaining sample plate
barcodes as prompted by the software.
For the tube of Kit Control DNA, manually enter the barcode you created
earlier.
B.
chapter 3 | Running the Protocol
The specific sample plate barcode to be scanned is displayed in this
field. When more than one sample plate is being used, you must scan
the sample plates in the order prompted by the software. In this
example, SMP2_0000601 had to be scanned first. After pressing Enter,
the software prompts the user to scan the barcode of sample plate
SMP2_wo0260_KitControl.
47
Status Ready = a transfer between
a source plate and a destination
(plate or array) has been assigned
in the software. The transfer can
now be physically performed in the
lab. Transfers with the status
Ready can be edited (deleted, for
example).
Barcodes scanned
or entered manually
appear here.
Figure 3.27
Run Anneal Plate Window – Scanning Sample Plate Barcodes
Scan the Anneal Plate barcode and press Enter or click Next.
F. Scan the protocol barcode and press Enter or click Next.
A list of available protocols is displayed in the upper part of the window.
G. In the Thermal Cycler field, type the number of the thermal cycler the plate
will be annealed on and press Enter or click Next.
H. Click Save; then click Close.
Notice that the name of the Anneal Plate in the left pane is now the barcode that you
scanned. In this example, it changed from Anneal Plate:4 to ANN_0000201.
Information about the Anneal Plate is also now displayed in the right pane under the
Properties tab (the Anneal Plate name must be selected to see this information; Figure
3.28).
E.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Figure 3.28
Anneal Plate Properties
ALIQUOT SAMPLES FROM SAMPLE TO ANNEAL PLATES
To aliquot samples from a sample plate to an Anneal Plate:
Spin down the sample plates at 1000 rpm for 30 seconds.
2. Place the sample and Anneal Plates in plate holders.
3. Spin down the Kit Control DNA.
4. In GTGS, select the Anneal Plate and click the Incoming Transfers tab in the
right pane.
Notice that the Status of each transfer has changed to Complete (Figure 3.29.)
Once the barcode of the destination plate or array is scanned, the software assumes
that the samples have been physically transferred from the source to the
destination. You cannot edit transfers with a status of Complete.
1.
5.
Transfer samples from the designated sample plate wells (Source and Source
Location) to the Anneal Plate (Current and Current Location.)
• For 3K and 5K assay panels, aliquot 13.4 μL of:
- Genomic DNA sample to wells 1 through 11 of rows A and C, and wells
1 through 12 of rows B and D.
- Kit Control DNA to well 12 of rows A and C.
• For 10K and 20K assay panels, aliquot 26.7 μL
- Genomic DNA sample to wells 1 through 11 of rows A and C, and wells
1 through 12 of rows B and D.
- Kit Control DNA to well 12 of rows A and C.
chapter 3 | Running the Protocol
49
In this example, sample from well C12 of Sample Plate SMP2_000060 is to
be transferred to well B1 of Anneal Plate ANN_2222. Samples C1, 2, 3, 4, 5,
6, 7, 8, 9, 10 and 11 are to be transferred to wells A1, 2, 3, 4, 5, 6, 7, 8, 9, 10
and 11, and so on.
Kit Control DNA from the tube called SMP2_wo0260_KitControl is to be
aliquoted to wells A12 and C12 of the Anneal Plate ANN_2222.
The assay panel to be used to prepare the Anneal Cocktail is also listed
(5-0059 3K Training Assay Panel in this example.) The Anneal Cocktail is to
be aliquoted to all wells being used on the Assay Plate.
Status Complete = the Anneal Plate barcode has been scanned and the
samples have been transferred from the Sample to the Anneal Plate.
Transfers with the Status Complete cannot be changed.
Figure 3.29
Sample Plate to Anneal Plate Transfers
PREPARING SPECIAL EDITION ASSAY PANELS
If you are working with a Special Edition assay panel, then your GeneChip SNP Kits
contain two tubes of assay panel mix: one tube containing the Standard Panel mix (fill
size 160 μL), and one tube containing the Supplemental Panel mix (fill size 60 μL).
Before proceeding to the next step, Prepare Anneal Cocktail , you must combine the
contents of these tubes as directed below.
To prepare the Special Edition assay panel:
1.
Combine the standard and supplemental assay panels as follows:
To a 1.5 mL Eppendorf tube, add the appropriate amount of standard assay
panel as shown in Table 3.2.
B. Rinse the pipette tip by pipetting up and down 5 times.
C. Add the appropriate amount of supplemental assay panel as shown in Table 3.2.
D. Rinse the pipette tip by pipetting up and down 5 times.
2. Cap the tube and spin down.
3. Mix by vortexing.
4. Spin down again and place on ice until ready to use.
A.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
The combination of Standard and Supplemental assay panels is now referred
to as the Assay Panel.
Table 3.2
Preparing the Special Edition Assay Panel Mix
Tube
1 Reaction
24 Reactions
(20% extra)
48 Reactions
(20% extra)
Standard Assay Panel Tube
(fill size 160 µL)
3.75 µL
144 µL
288 µL
Supplemental Assay Panel Tube
(fill size 60 µL)
1.25 µL
48 µL
96 µL
5 µL
192 µL
384 µL
Total
PREPARE ANNEAL COCKTAIL
Enzyme A is extremely temperature sensitive. Keep at –20°C until ready to use, and
add last to the cocktail. To avoid denaturing, keep the Anneal Cocktail on ice until
ready to use. Minimize warming by hand contact.
To prepare the Anneal Cocktail:
Label a 2 mL Eppendorf tube with the letters AC.
2. Vortex and spin down Buffer A and the Assay Panel.
If using a Special Edition assay panel, the contents of the Standard and
Supplemental assay panel tubes should have already been combined. See Preparing
Special Edition Assay Panels on page 49.
3. Place on ice until ready to use.
4. To the tube labeled AC, add and mix the reagents listed in Table 3.3 in the order
shown.
1.
Thaw and spin down Enzyme A before adding to the cocktail.
Store the remaining Water at 4°C.
6. Using a P1000 single-channel pipette, mix the Anneal Cocktail by pipetting up
and down 5 times.
7. Keep on ice until ready to use.
5.
chapter 3 | Running the Protocol
51
Table 3.3
Anneal Cocktail
Reagents
1 Reaction
2 Rows
(20% extra)
4 Rows
(20% extra)
Water (from kit) (3K/5K blue; 10K/20K green)
21.6 µL
8.3 µL
622 µL
239 µL
1244 µL
478 µL
Buffer A
5.00 µL
144 µL
288 µL
Assay Panel
5.00 µL
144 µL
288 µL
Enzyme A (keep at -20°C until addition)
0.0625 µL
1.8 µL
3.6 µL
Total Volume Anneal Cocktail
(3K/5K blue; 10K/20K green)
31.6 µL
18.3 µL
912 µL
529 µL
1824 µL
1058 µL
ALIQUOT ANNEAL COCKTAIL AND BEGIN INCUBATION
To aliquot the Anneal Cocktail and begin incubation:
Place the Anneal Plates on ice.
2. If using manual 24-channel pipettes, add the Anneal Cocktail as follows:
1.
Place a 50 mL reagent reservoir on ice.
B. Pipette the Anneal Cocktail into the 50 mL reagent reservoir.
C. Aliquot the Anneal Cocktail as follows (Table 3.4):
- For 3K/5K assay panels: Use a 12-channel P200 to add 31.6 μL to each reaction
- For 10K/20K assay panels: Use a 12-channel P20 to add 18.3 μL to each
reaction
D. Set a 24-channel P100 to 30 μL, and mix each sample by pipetting up and
down 10 times.
A.
3.
If using electronic 24-channel pipettes, add the Anneal Cocktail as follows:
A.
Aliquot the cocktail to two strips of 12 tubes each as follows:
- For 3K/5K assay panels: Use a single-channel P200 to aliquot 35 μL (70 μL) to
each tube
- For 10K/20K assay panels: Use a single-channel P20 to aliquot 20 μL (40 μL)
to each tube
B.
Aliquot the cocktail to each reaction by dispensing/mixing 10 times for each
reaction as follows (Table 3.4):
- 3K/5K assay panels: Use a P200 set to dispense/mix at 31.6 μL.
- 10K/20K assay panels: Use a P20 set to dispense/mix at 18.3 μL.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
.
Table 3.4
Genomic DNA, Kit Control DNA and Anneal Cocktail
Component
3K and 5K Assay
Panels
10K and 20K Assay
Panels
Genomic DNA (150 ng/µL) or Kit Control DNA
13.4 µL
26.7 µL
Anneal Cocktail
31.6 µL
18.3 µL
Total
45 µL
45 µL
4.
5.
6.
7.
8.
Seal each plate with a MicroAmp adhesive film, covering all wells even if empty.
Spin down the Anneal Plate at 1000 rpm for 30 seconds.
Start the thermal cycler program, Meg Anneal (Figure 3.30).
When the temperature on the thermal cycler reaches 20°C, load the Anneal Plate
and close the lid.
Incubate the samples for 16 to 24 hours.
Optimal incubation time is 16 to 24 hours. Do not incubate samples for more than
30 hours.
Figure 3.30
Meg Anneal Thermal Cycler Program
PREPARE FOR STAGE 4
To prepare for stage 4:
Place three to four 96-well aluminum blocks in a -20°C freezer overnight.
chapter 3 | Running the Protocol
53
Stage 3 – Plan and Run Assay Plates
ABOUT THIS STAGE
During this stage, you will plan and prepare what are referred to as Assay Plates. An
Assay Plate is a 96-well PCR plate with the barcode designation ASY<barcode>. You
will use GTGS to perform this step.
LOCATION AND DURATION
• Pre-Amp Lab
• Hands-on time: approximately 10 minutes
EQUIPMENT AND MATERIALS REQUIRED
The following materials are required to perform this stage.
Table 3.5
Quantity
Item
1
Marking pen, extra fine point, permanent
2
PCR plate, 96-well, with ASY barcode
PLAN ASSAY PLATES
To plan Assay Plates:
1.
Using a permanent marking pen, label the ASY-barcoded PCR plates as shown in
Figure 3.31.
One Assay Plate can hold up to two full rows of samples from an Anneal Plate.
Samples are placed in rows A and E only of the Assay Plate.
To ensure the highest quality data, we strongly recommend running one positive
control for every 23 samples. Control DNA is included in each GeneChip SNP Kit.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
SAMPLES
X
A
C
Anneal Plate row
designations.
G
T
X
A
C
G
T
Figure 3.31
Assay Plate Labeling
In GTGS, expand the Project and Tracking icons.
3. Right-click the Assay Plates icon and select Add Assay/Label/Hyb Plate Trio.
4. Open the Source menu and select an Anneal Plate.
You cannot change the Destination (Assay Plate).
5. Select the samples to be transferred from the Anneal Plate to the Assay Plate using
one of these methods:
• Highlight a row of samples on the Anneal Plate.
• Select an individual sample on the Anneal Plate, such as sample B3 only.
• Select a smaller group of samples, such as samples B1 through B4 only.
2.
6.
Do one of the following:
• If you selected an entire row, highlight row A or E on the Assay Plate, then click
Set (see Figure 3.32.)
• If you selected one sample, highlight one well in row A or E on the Assay Plate,
then click Set.
• If you selected a smaller group of samples, highlight the corresponding number
of cells in row A or E on the Assay Plate, then click Set.
Each Assay Plate can hold a total of 24 reactions (including controls). When
planning the Assay Plate in GTGS, the software restricts you to mapping samples
from the Anneal Plate to rows A and E only of the Assay Plate.
7.
When all sample transfer designations are complete, click Save.
chapter 3 | Running the Protocol
55
Row A on Anneal
Plate designated for
transfer to Row A of
Assay Plate.
Row B on Anneal
Plate designated for
transfer to Row E of
Assay Plate.
After clicking Set,
sample indicators
change to color
code for Pending.
Figure 3.32
Planning an Assay Plate
Expand the Assay, Label and Hyb Plates icons. Notice there is now a placeholder for
each type of plate in these folders. These three plates are linked. As you move through
the protocol, each one will automatically appear in the plate mapping Source or
Destination field as appropriate. Assay Plate samples are transferred to Label Plates,
and Label Plate samples are transferred to Hyb Plates.
To delete an Assay/Label/Hyb plate trio, you must select and delete the Hyb Plate.
This action deletes the associated Assay and Label Plates as well. Once you begin
the next stage of the protocol and scan an Assay Plate barcode, you can no longer
delete these plates.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
RUN ASSAY PLATES
To run Assay Plates:
1.
2.
3.
4.
5.
6.
7.
Expand the Assay Plates icon.
Right-click an Assay Plate and select Run Assay Plate.
Scan the Anneal Plate barcode (appears in the Source field) and press Enter.
Scan the Assay Plate barcode (appears in the Source field) and press Enter.
Press Enter again to proceed past the protocol barcode which is already displayed
in the Protocol field.
Enter a thermal cycler number and press Enter.
Click Save; then click Close.
Notice that information about the Assay Plate is now displayed in the right pane
under each of the tabs (Figure 3.33).
Properties tab for the Assay Plate.
Incoming Transfers tab
Outgoing Transfers tab
Well Status tab
Figure 3.33
Assay Plate Tabs
chapter 3 | Running the Protocol
57
Stage 4 – Gap Fill, dNTP, Ligate, Invert, First PCR
ABOUT THIS STAGE
During this stage, samples are transferred from the Anneal Plate to two Assay Plates.
During the transfer, samples are split into four equal aliquots. Several additions are
then made to each sample, one addition at a time, at specific intervals. The additions
designated as cold are made on ice; the additions designated as hot are made while the
samples are on a thermal cycler.
The thermal cycler program run for these additions is determined by the size of the
assay panel you are using. It will be one of the following: the Meg 3K 5K 10K program,
or the Meg 20K program.
Gap Fill Mix Addition
The first addition, Gap Fill Mix, is a cold addition. Gap fill enzymes find and bind to
the single base gap in the assay panel probe. The gap is centered where the SNP of
interest is located in the genome.
Gap Fill Enzyme
Figure 3.34
Gap Fill Enzyme Addition
dNTP Addition & Ligation
Each sample is now split into 4 equal aliquots in the Assay Plate, followed by the
addition of dNTPs. For this cold addition, a different nucleotide is added to each
aliquot. In the example shown in Figure 3.35, only the aliquot containing dATP will
undergo a reaction, wherein the gap fill enzymes will use dATP to fill the gap in the
probe.
This example is based on a homozygous SNP locus. If the sample was
heterozygous for this locus, two of the four aliquots would have a nucleotide added
to the probe.
Once the gap in the probe is filled, the backbone of the assay probe is covalently sealed.
The sealed probe is now referred to as a padlocked probe.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Only the probe in the A
aliquot has a
nucleotide added to it.
The gap in the G, C and
T aliquots is never
filled; these probes
remain linear.
Figure 3.35
dNTP Addition
This example depicts one assay probe only. In reality, thousands of probes are undergoing the
same process simultaneously.
Exo Mix Addition
The third addition is the Exo Mix, a cold addition. In this step, exonucleases are added
to each aliquot. The exonucleases digest the linear probes and single-stranded DNAs
that are present (Figure 3.36). Linear probes are present in the aliquots where the gap
was not filled by a dNTP. Single-stranded DNA is present in all aliquots.
Figure 3.36
Exo Mix Addition Showing Two Aliquots Only
chapter 3 | Running the Protocol
59
For homozygotes:
After the Exo Mix addition, three of the four aliquots contain digested DNA only
for each assay probe. In the fourth aliquot, the genomic DNA is mainly digested
away except for the small region where it is double-stranded due to its association
with the padlocked probe.
For heterozygotes:
After the Exo Mix addition, two of the four aliquots contain digested DNA only.
In the other two aliquots, the genomic DNA is mainly digested away except for
the small region where it is double-stranded due to its association with the
padlocked probe.
Cleavage Mix Addition and Inversion
The fourth addition is the Cleavage Mix, a hot addition. The padlocked probe is
cleaved at cleavage site 1 in the assay probe backbone, thereby releasing it from the
genomic DNA.
Cleavage site 1
Figure 3.37
Cleavage Mix Addition
Once the assay probe has been cleaved, it releases from the remaining genomic DNA
and becomes a linear molecule. Because the orientation of the PCR primer sequences
has changed from the original orientation, the probe is now referred to as an inverted
probe.
After cleavage and release from the genomic DNA, the probe inverts.
Figure 3.38
Inverted Probe
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Amp Mix Addition and First PCR
The fifth addition is the Amp Mix, a hot addition. Once the Amp Mix is added, a PCR
reaction takes place and the probe is amplified using common primers. The product
from this reaction is referred to as the first PCR product (Figure 3.39).
Figure 3.39
Amplified Probes from First PCR
LOCATION AND DURATION
• Pre-Amp Lab
• Hands-on time: 2.5 hours
EQUIPMENT AND MATERIALS REQUIRED
The following equipment and materials are required to perform these stages.
Quantities shown are for processing 48 reactions.
Table 3.6
Equipment and Materials Required for Stage 4 – Gap Fill, dNTP, Ligate, Invert, First PCR
Quantity
3 or 4
Item
Aluminum blocks, chilled (or 2 aluminum blocks and 1 to 2 plate holders)
1
Anneal Plate from previous stage
2
Assay Plates, prepared and scanned
1
Centrifuge, plate
1
Ice container, rectangular, filled with ice
12
MicroAmp Clear Adhesive Films
1
Microfuge
2
Reagent reservoirs, 50 mL
2
Thermal cyclers, 96-well GeneAmp PCR System 9700
4
Tube strips with caps, PCR 12-well
chapter 3 | Running the Protocol
61
KIT COMPONENTS REQUIRED
The following components are required from each GeneChip SNP Kit:
• Gap Fill Mix
• Exo Mix
• Cleavage Enzyme
• Cleavage Tube
• Amp Mix
• Plate of dNTPs (located in compartment below reagent tubes in the Pre-Amp Lab
box)
OTHER REAGENTS REQUIRED
Stratagene Taq Polymerase (P/N 929197), 134 μL.
For more information, refer to Taq Polymerase Required on page 7.
THAW THE REAGENTS
To thaw the reagents:
Thaw the plate of dNTPs, the Cleavage Tube, and Amp Mix on the bench top.
2. Keep on ice until ready to use.
1.
Leave the Exo Mix, Gap Fill Mix, Cleavage Enzyme, and Taq Polymerase at -20°C
until ready to use.
PREPARE THE GAP FILL MIX, EXO MIX, AND DNTP PLATE
Volumes shown are for 24 reactions (and 48 reactions) respectively.
Prepare the Gap Fill Mix
To prepare the Gap Fill Mix:
Place a plate holder or a chilled aluminum block on ice.
2. Spin down the Gap Fill Mix (G).
3. Label two strips of 12 PCR tubes with the letter G.
4. Aliquot 5 μL (10 μL) of Gap Fill Mix to each tube.
1.
Cap and spin down the strip tubes.
6. Place the tubes in a plate holder or chilled aluminum block in adjacent rows.
7. Keep on ice until ready to use.
5.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Prepare the Exo Mix
To prepare the Exo Mix:
1.
2.
3.
4.
5.
Spin down the Exo Mix (E).
Label two strips of 12 PCR tubes with the letter E.
Aliquot 21 μL (42 μL) of Exo Mix to each tube.
Cap and spin down the strip tubes.
Place the tubes in the same plate holder or chilled aluminum block as the Gap Fill
Mix.
Be sure to keep the two mixes separated and easily identifiable. These mixes are
added to the samples at different times. The Gap Fill Mix is added first.
To save time, you can prepare the Exo Mix during the first or second 58°C
incubation.
Prepare the dNTP Plate
To prepare the dNTP Plate:
Spin down the plate of dNTPs.
2. Place on ice until ready to use.
1.
ADD GAP FILL MIX – ADDITION #1 ON COLD BLOCK
To add the Gap Fill Mix:
These instructions are based on working with 48 reactions.
Remove two aluminum blocks from the −20°C refrigerator and set on ice.
2. Stop the Meg Anneal thermal cycler program.
3. Remove the Anneal Plate from the thermal cycler.
1.
4.
5.
6.
7.
8.
Place the Anneal Plate in a chilled aluminum block on ice and cool for 2 min.
Spin down the plate at 1000 rpm for 30 seconds.
Place an Assay Plate in an aluminum block on ice.
Slowly remove the clear film from the Anneal Plate.
Working two rows at a time on the Anneal Plate (Figure 3.40), add the Gap Fill
Mix using one of the following methods:
• If using manual 24-channel pipettes:
Use a P20 to add 2.5 μL of Gap Fill Mix to each sample.
2) Mix up and down 3 times to rinse all of the Gap Fill Mix from the pipette
tip.
3) Set a P200 to 20 μL and mix each sample up and down 10 times.
1)
chapter 3 | Running the Protocol
63
• If using electronic 24-channel pipettes:
Set a P20 to dispense at 2.5 μL and mix at 19 μL.
2) Dispense/mix Gap Fill Mix to each sample 10 times.
1)
24-channel pipette
Anneal Plate in
aluminum block on ice
Figure 3.40
Cold Addition
TRANSFER AND SPLIT SAMPLES
You will now transfer one row of samples from the Anneal Plate to four rows of an
Assay Plate, splitting each sample into four aliquots of equal size.
To transfer and split samples:
1.
Using a 12-channel P10 pipette, transfer and split the samples from the first two
rows of the Anneal Plate to an Assay Plate as follows (Figure 3.41):
A.
Transfer 9 μL from each well of the first row (A1 to A12 in the example) of the
Anneal Plate to the rows marked A, C, G and T on the top half of the Assay
Plate (A1 to D12.)
B.
Transfer 9 μL from each well of the next row (B1 to B12 in the example) of the
Anneal Plate to the rows marked A, C, G and T on the bottom half of the Assay
Plate (E1 to H12.)
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Samples from row A of the Anneal Plate are transferred and split between rows A, B, C and D (marked A, C, G and T) of
the first Assay Plate. Samples from row B of the Anneal Plate are transferred and split between rows E, F, G and H of the
first Assay Plate. C = Kit Control DNA.
NOTE: The samples in rows C and D are transferred to the second Assay Plate and are split in the same manner.
SAMPLES
SAMPLES
C X
C A
X
C C
X
C X
C G
X
C
X
A
C
X
X
T
X
X
G
T
Figure 3.41
Transferring Samples from the Anneal Plate to the Assay Plates
2.
3.
4.
5.
6.
7.
Seal all of the wells of the Assay Plate with clear adhesive film.
Spin down the Assay Plate at 1000 rpm for 30 seconds.
Place in an aluminum block on ice.
Start the appropriate thermal cycler program:
• For 3K/5K/10K assay panels: Meg 3-5-10K
• For 20K assay panels: Meg 20K
See Figure 3.42 on page 65.
When the thermal cycler reaches 58°C, load the Assay Plate and close the lid.
For 48 reactions, repeat the transfer and split process for the remaining two rows
of samples on the Anneal Plate to the second Assay Plate plate.
The placement of each Assay Plate onto a thermal cycler should be staggered by
approximately 2 minutes.
8.
Leave the aluminum blocks on ice.
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65
Figure 3.42
Meg 3-5-10K and Meg 20K Thermal Cycler Programs
ADD DNTPS – ADDITION #2 ON COLD BLOCK
To add the dNTPs:
Perform this procedure one Assay Plate at a time.
Per
1.
When the thermal cycler timer reads 1:00 minute remaining for the second 58°C
period (Figure 3.42), press Pause.
2.
Remove the Assay Plate and place it in an aluminum block on ice for 2 minutes.
PHYSICAL INJURY HAZARD. The thermal cycler lid, sample block and Assay Plate
are very hot. Wear protective gloves when opening and closing the lid, and when
handling Assay Plates.
Remove the seal from the Assay Plate and the dNTP plate.
4. If using a manual 24-channel P20 pipette, add dNTPs to each reaction as follows
(Figure 3.43):
3.
Aliquot 4.0 μL dATP to row A, and 4.0 μL dCTP to row B.
B. Mix by pipetting up and down 20 times, reaching the bottom of all wells.
C. Aliquot 4.0 μL dGTP to row C, and 4.0 μL dTTP to row D.
D. Mix by pipetting up and down 20 times, reaching the bottom of all wells.
5. If using an electronic 24-channel P20 pipette, add dNTPs to each reaction as
follows (Figure 3.43):
A.
Set the pipette to dispense and mix at 4 μL.
B. Dispense/mix dNTPs to each sample 20 times.
6. Repeat these additions for the bottom half of the plate.
A.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
SAMPLES
X
X
C A
dATP addition
C C
dCTP addition
C G
dGTP addition
C T
dTTP addition
A
dATP addition
C
dCTP addition
G
dGTP addition
T
dTTP addition
Figure 3.43
dNTP Addition
Reseal the plate with a new adhesive film.
8. Spin down at 1000 rpm for 30 seconds.
9. Place the plate back on the thermal cycler and close the lid.
10. Press Resume to continue the thermal cycler program.
11. Repeat this procedure for all remaining Assay Plates.
7.
ADD EXO MIX – ADDITION #3 ON COLD BLOCK
To add the Exo Mix:
Perform this procedure one Assay Plate at a time.
1.
When the timer reads 14:00 minutes remaining for the first 37°C period
(Figure 3.42 on page 65):
A.
Press Pause on the thermal cycler.
Remove the Assay Plate and place in an aluminum block on ice for 2 minutes.
C. If using a manual 24-channel P20 pipette:
B.
1)
Add 4 μL of Exo Mix to each reaction.
Mix by pipetting up and down 20 times, reaching the bottom of all wells.
D. If using an electronic 24-channel P20 pipette:
2)
Set the pipette to dispense and mix at 4 μL
2) Add Exo Mix to each reaction, mixing each reaction 20 times.
Reseal the plate with a new adhesive film.
Spin down at 1000 rpm for 30 seconds.
Place the plate back on the thermal cycler and close the lid.
Press Resume to continue the thermal cycler program.
Repeat this procedure for all remaining Assay Plates.
1)
2.
3.
4.
5.
6.
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67
PREPARE THE CLEAVAGE AND AMP MIXES
Prepare the Cleavage Mix
Prepare during first 95°C hold.
To prepare the Cleavage Mix:
1.
2.
3.
4.
5.
Spin down the Cleavage Enzyme.
Using a P200 single channel pipette, add 24 μL of Cleavage Enzyme to each
Cleavage Tube.
This mixture is now referred to as the Cleavage Mix.
Using a P1000 single channel pipette, mix well.
Mark a 50 mL reagent reservoir with the letter C.
Transfer the Cleavage Mix to the reagent reservoir marked C and place on ice.
Prepare the Amp Mix
Prepare during first 95°C hold.
To prepare the Amp Mix:
1.
2.
3.
4.
5.
pin down the Stratagene Taq polymerase.
Using a P200 single channel pipette, add 67 μL of Taq polymerase to the Amp Mix
tube.
Using a P1000 single channel pipette, mix well.
Mark a 50 mL reagent reservoir with the letter A.
Transfer the Amp Mix to the reagent reservoir marked A and place on ice.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
ADD CLEAVAGE MIX – ADDITION #4 ON THERMAL CYCLER
Perform this addition one Assay Plate at a time.
To add the Cleavage Mix:
1.
When the thermal cycler timer reads 9:00 minutes remaining for the second 37°C
period (Figure 3.42 on page 65):
Press Pause on the thermal cycler and open the lid.
B. Leaving the Assay Plate on the cycler, remove the adhesive film.
C. Using a 12-channel P200 pipette, aliquot 25 μL of Cleavage Mix to each
reaction.
A.
Mix by pipetting up and down 10 times (Figure 3.44).
E. Reseal the plate with a new adhesive film, and close the thermal cycler lid.
F. Press Resume to continue the thermal cycler program.
2. Repeat this procedure for all remaining Assay Plates.
D.
Figure 3.44
Hot Addition on Thermal Cycler
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69
ADD AMP MIX – ADDITION #5 ON THERMAL CYCLER
Perform this addition one Assay Plate at a time.
To add the Amp Mix:
1.
When the thermal cycler temperature reaches 60°C (Figure 3.42 on page 65):
Press Pause on the thermal cycler and open the lid.
B. Leaving the Assay Plate on the cycler, remove the adhesive film.
C. Using a 12-channel P200 pipette, aliquot 25 μL of Amp Mix to each reaction.
D. Mix by pipetting up and down 10 times.
A.
Reseal the plate with a new adhesive film.
F. Close the thermal cycler lid.
G. Press Resume to continue the thermal cycler program.
2. Repeat this procedure for all remaining Assay Plates.
E.
TRANSFER ASSAY PLATES AND KIT COMPONENTS TO POST-AMP LAB
To transfer the Assay Plates and remaining GeneChip SNP Kit components:
CONTAMINATION DANGER. Do not remove the adhesive film from the Assay
Plates. Assay Plates must remain sealed until they have been transferred to the
Post-Amp Lab.
At the end of the program (approximately one hour after the start of the first PCR
amplification), remove the Assay Plates from the thermal cyclers.
2. Leaving the plates sealed, transfer the plates and the remaining GeneChip SNP Kit
contents to the Post-Amp Lab.
3. Store the kit contents in the appropriate refrigerator or freezer.
4. If not immediately proceeding to Stage 5 – Second PCR, the Assay Plates can be
stored at -20°C.
1.
We recommend freezing and thawing plates ONE TIME ONLY. For a given set of
samples, do not freeze and thaw plates multiple times during the protocol.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Stage 5 – Second PCR
ABOUT THIS STAGE
During this stage, you will prepare what are referred to as Label Plates (96-well PCR
plates with the barcode designation LBL<barcode>.) Each sample is transferred from
the Assay Plate to a Label Plate.
After the transfer, a different allele-specific primer is added to each reaction as a label,
and the second PCR is performed. The thermal cycler program used is determined by
the size of the assay panel. For 3K and 5K assay panels, Meg Hypcr 3-5K is used. For
10K and 20K assay panels, Meg Hypcr 10-20K is used.
First PCR product
Tag
Second PCR product with
allele-specific labeling
Figure 3.45
Second PCR
LOCATION AND DURATION
• Post-Amp Lab
• Hands-on time: Approximately 25 minutes
• Thermal cycler time: 30 minutes
Amplified DNA
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71
EQUIPMENT AND MATERIALS REQUIRED
The following equipment and materials are required to perform this stage. Quantities
shown are for processing 48 reactions.
Table 3.7
Equipment and Materials Required for Stage 5 – Second PCR
Quantity
Item
2
Assay Plates with samples from previous stage
1
Centrifuge, plate
1
Ice container, rectangular, filled with ice
2
PCR plates, 96-well, with LBL barcode
2
Marking pens, extra fine point, permanent, different colors
minimum 4
MicroAmp Clear Adhesive Films
1
Microfuge
4
Plate holders (preparation racks)
4
Reagent reservoirs, 50 mL
2
Thermal cyclers
KIT COMPONENTS REQUIRED
The following components are required from each GeneChip SNP Kit:
• HY A Allele Tube
• HY C Allele Tube
• HY G Allele Tube
• HY T Allele Tube
OTHER REAGENTS REQUIRED
Clontech TITANIUM Taq DNA Polymerase
For more information, refer to Taq Polymerase Required on page 7.
THAW REAGENTS
To thaw reagents:
1.
Thaw the HY A Allele, HY C Allele, HY G Allele, and HY T Allele tubes on the
bench top.
Spin down each tube.
3. Keep on ice until ready to use.
2.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
RUN LABEL PLATES
To run Label Plates:
1.
Using a permanent marking pen, mark the LBL barcoded plates as shown in
Figure 3.46.
SAMPLES
C A
A Allele Tube Mix
C C
C Allele Tube Mix
C G
G Allele Tube Mix
C T
T Allele Tube Mix
A
A Allele Tube Mix
C
C Allele Tube Mix
G
G Allele Tube Mix
T
T Allele Tube Mix
Mx
Mx
Figure 3.46
Label Plate Labeling and Sample Placement
In GTGS, expand the Project, Tracking and Label Plate icons.
3. Right-click the appropriate Label Plate.
If you are not sure which Label Plate is correct, select a Label Plate. In the righthand pane, look at the Assay Plate indicated in the Source column under the
Incoming Transfers tab.
4. Select Run Label Plate.
The Run Label Plate window appears (Figure 3.47).
2.
Figure 3.47
Run Label Plate Window
H.
Scan the Assay Plate barcode (Source Plate field) and press Enter.
chapter 3 | Running the Protocol
73
Scan the Label Plate barcode (Label Plate field) and press Enter.
J. Press Enter again to proceed past the protocol barcode which is already
displayed in the Protocol field.
K. Enter a thermal cycler number and press Enter.
I.
Click Next; then click Save.
5. Using a marking pen, mark the Assay and Label Plates with the same color.
Use a different color for each set of plates to ensure that samples are transferred
from each Assay Plate to the correct Label Plate.
6. Repeat these steps for the next Label Plate.
L.
PREPARE HY ALLELE TUBE MIXES
To prepare the Allele Tube Mixes:
1.
2.
3.
4.
5.
Spin down the TITANIUM Taq polymerase.
Using a P100 pipette, add 22 μL of Taq polymerase to each allele tube.
Set a P1000 pipette to 750 μL and mix by pipetting up and down 10 times.
Label one set of 12 strip tubes for each HY Allele mix: A, C, G and T
Aliquot 75 μL (150 μL) of each allele tube mix as follows:
• HY A Allele Tube Mix to the strip tubes labeled A.
• HY C Allele Tube Mix to the strip tubes labeled C.
• HY G Allele Tube Mix to the strip tubes labeled G.
• HY T Allele Tube Mix to the strip tubes labeled T.
TRANSFER REACTIONS TO LABEL PLATES AND ADD ALLELE TUBE MIXES
To transfer reactions to Label Plates and add Allele Tube mixes:
Working one row at a time using a 12-channel P200 pipette, aliquot 31 μL of each
Allele Tube mix to the corresponding row on the Label Plate (Figure 3.44):
• HY A Allele Tube mix to rows labeled A (A and E)
• HY C Allele Tube mix to rows labeled C (B and F)
• HY G Allele Tube mix to rows labeled G (C and G)
• HY T Allele Tube mix to rows labeled T (D and H)
2. If using manual 24-channel pipettes:
1.
Set a P20 to 4 μL and transfer 4 μL from each well of the top two rows of the
Assay Plate (marked A and C) to the same rows on the Label Plate.
B. Mix by pipetting up and down 5 times.
C. Set a P100 to 20 μL, and mix by pipetting up and down 10 times.
D. Continue transferring and mixing the remaining reactions from the Assay Plate
to the Label Plate in the same manner.
A.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
If using electronic 24-channel pipettes:
Set a P20 to dispense/mix at 4 μL.
B. Dispense/mix 4 μL from each well of the top two rows of the Assay Plate
(marked A and C) to the same rows on the Label Plate 5 times.
C. Set a P100 set to dispense/mix at 20 μL. Mix each reaction by dispensing/
mixing 10 times.
D. Continue transferring and mixing the remaining reactions from the Assay Plate
to the Label Plate in the same manner.
Seal each Label Plate with a new adhesive film.
Spin down the Label Plates at 1000 rpm for 30 seconds.
Place each Label Plate onto a thermal cycler and close the lid.
Start the appropriate thermal cycler program:
A.
4.
5.
6.
7.
• For 3K/5K assay panels, use Meg Hypcr 3-5K
• For 10K/20K assay panels, use Meg Hypcr 10-20K (Figure 3.48).
This program takes approximately 30 minutes to run.
Figure 3.48
Meg Hypcr 3-5K and Meg Hypcr 10-20K Thermal Cycler Programs
If running a quality control gel, refer to First Quality Control Gel on page 75 now.
9. When the program is finished, remove the plates and spin down at 1000 rpm for
30 seconds.
10. Proceed directly to Stage 6 – Target Digest on page 77.
8.
Do not freeze samples at this stage of the protocol. Proceed directly to the next
stage.
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75
FIRST QUALITY CONTROL GEL
The first quality control gel is run while the second PCR thermal cycler program is
running (Meg Hypcr 3-5K or Meg Hypcr 10-20K). Samples are taken from the Assay
Plates. For gel recommendations, see Quality Control Gels on page 19.
To run the first quality control gel:
1.
2.
3.
4.
5.
6.
Pipette 7 μL of each sample from an Assay Plate to a 96-well PCR plate.
Add 3 μL loading buffer and mix well.
Load onto a gel.
Repeat steps 1 through 3 for each Assay Plate and gel.
Seal the Assay Plates with new adhesive films and store at -20°C.
Run and inspect the gels.
VIEWING THE FIRST QUALITY CONTROL GEL
A good first quality control gel will look like the one shown below.
For this sample, differences in band intensity indicate that there is variation in the amount of product in each well.
This amount of variation is typically acceptable. See
Figure 3.50 for an example of unacceptable well variation.
More product can appear
in the center wells than
in the side wells.
AC
GT
Figure 3.49
Good First Quality Control Gel for Assay Panel run on a 10K Universal Tag Array – Top Half Only
Positive controls look
identical in appearance
and number of bands as
compared to samples.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Unacceptable well variation. These samples
failed when hybridized to arrays and
scanned.
Failure was due to unacceptably low signal
strength in several channels. Repeat the
experiment from Stage 1.
Figure 3.50
First Quality Control Gel with Mixed Results
Samples in wells 17 through 22 passed
when hybridized to arrays and scanned.
The variation in appearance of these
bands is within acceptable limits.
chapter 3 | Running the Protocol
77
Stage 6 – Target Digest
ABOUT THIS STAGE
During this stage, the labeled second PCR product is cleaved at cleavage site 2 in the
backbone. This process removes the amplified genomic DNA portion of the assay
probe from the tag and allele-specific label.
As part of this stage, you will prepare what are referred to as Hyb Plates (96-well PCR
plates with the barcode designation HYB<barcode>).
You will then transfer the four individual reactions for each sample from the Label
Plate to a Hyb Plate. At the same time, you will consolidate the four reactions for each
sample back to one well. The Hyb Plates are then placed on thermal cyclers and the
Meg Hydigest-a program is run.
First PCR product
Tag
Second PCR product – allelespecific labeling
Cleave amplified DNA from probe at cleavage
site 2 to release allele-specific products
Figure 3.51
Target Digest
LOCATION AND DURATION
• Post-Amp Lab
• Hands-on time: approximately 30 minutes
• Time on thermal cycler: 1 hour and 35 minutes
Amplified DNA
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
EQUIPMENT AND MATERIALS REQUIRED
The following equipment and materials are required to perform this stage. Quantities
shown are for processing 48 reactions.
Table 3.8
Quantity
Item
1
Centrifuge, plate
1
Ice container, rectangular, filled with ice
2
Label Plates containing samples from previous stage
1
Marking pen, extra fine point, permanent
4
MicroAmp Clear Adhesive Films
1
Microfuge
2
PCR plates, 96-well, with HYB barcode
3
Plate holders (preparation racks)
2
Thermal cyclers
2
Tube strip with caps, PCR 12-well
PHYSICAL INJURY HAZARD. The thermal cycler lid, sample block and PCR plate are
very hot. Wear protective gloves when opening and closing the lid, and when
handling the PCR plate.
KIT COMPONENTS REQUIRED
The following component is required from each GeneChip SNP Kit:
• HY Digest Mix
RUN HYB PLATES
To run Hyb Plates:
1.
Using a permanent marking pen, label the HYB-barcoded plates as shown in
Figure 3.52 on page 79.
2.
In GTGS, expand the project, Tracking and Hyb Plates icons.
Right-click the appropriate Hyb Plate and select Run Hyb Plate.
To determine the correct Hyb Plate, select a Hyb Plate and click the Incoming
Transfers tab. Look at the Label Plate specified in the Source field.
Scan the Label Plate barcode (Source Plate field) and press Enter.
Scan the Hyb Plate barcode (Hyb Plate field) and press Enter.
Press Enter again to proceed past the protocol barcode which is already displayed
in the Protocol field.
3.
4.
5.
6.
chapter 3 | Running the Protocol
79
Enter a thermal cycler number and press Enter.
8. Click Save; then click Close.
7.
SAMPLES
C
Rows A through D of the Label
Plate are consolidated to row A of
the Hyb Plate.
Rows E through H of the Label
Plate are consolidated to row B of
the Hyb Plate.
Figure 3.52
Hyb Plate Labeling and Sample Placement
TRANSFER REACTIONS TO HYB PLATES
While transferring reactions from Label to Hyb Plates, you will consolidate all four
reactions for each sample into one well.
To transfer reactions to Hyb Plates:
Spin down each Label Plate at 1000 rpm for 30 sec.
2. Set a 12-channel P20 pipette to 17 μL.
3. Remove the seal from each Label Plate.
4. Transfer 17 μL from each well of Label Plate rows A, B, C and D to Hyb Plate
row A (Figure 3.52).
5. Transfer 17 μL from each well of Label Plate rows E, F, G and H to Hyb Plate
row B.
Total volume in each well should be 68 μL.
1.
ADD HY DIGEST MIX
Volumes shown are for 24 reactions (and 48 reactions) respectively.
To add the HY Digest Mix:
1.
2.
3.
4.
5.
Spin down the HY Digest Mix and keep on ice until ready to use.
Into two strips of 12 PCR tubes each, aliquot 9 μL (18 μL) of HY Digest Mix to
each tube using a P20 single channel pipette.
Cap and spin down the strip tubes.
Place the tubes in a plate holder on ice (in 2 adjacent rows) until ready to use.
If using manual 24-channel pipettes:
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Set a P20 to 6 μL.
B. Aliquot 6 μL of HY Digest Mix to each sample, and mix by pipetting up and
down 5 times to ensure the tip is thoroughly rinsed.
C. Set a P100 to 50 μL, and mix each reaction by pipetting up and down 15 times.
A.
6.
If using electronic 24-channel pipettes:
Set a P20 to dispense/mix at 6 μL.
B. Dispense/mix 6 μL of HY Digest Mix to each sample 5 times.
C. Set a P100 to dispense/mix at 50 μL.
D. Mix each sample by dispensing/mixing 15 times.
A.
The full volume must be picked up and dispensed each time to ensure that all of
the HY Digest Mix is flushed from the pipette tip.
Seal each plate with a new adhesive film.
8. Spin down at 1000 rpm for 30 seconds.
7.
RUN THE MEG HYDIGEST-A PROGRAM
To run the Meg Hydigest-a program:
Place each plate on a thermal cycler and close the lid.
2. Start the program, Meg Hydigest-a (Figure 3.53).
3. If running a second quality control gel, see Second Quality Control Gel on page 81.
You will remove sample from the plate at a specific point during the thermal cycler
program.
1.
4.
When the program is finished, remove the plates and proceed immediately to Stage
7 – Sample Hybridization on page 84.
Do not freeze samples at this stage of the protocol. Proceed directly to the next
stage.
If running a second QC gel, remove 4.0 μL
of each sample when the timer reads 5:00
remaining for the second 37°C hold.
Figure 3.53
Meg Hydigest-a Thermal Cycler Program
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81
SECOND QUALITY CONTROL GEL
This operation should be performed quickly and should not exceed 5 minutes.
To run a second quality control gel:
1.
2.
3.
4.
5.
When the timer reads 5:00 minutes remaining for the second 37°C hold
(Figure 3.53), press Pause.
Remove the plate from the thermal cycler and place on the bench top.
Remove the adhesive film from the plate.
Remove 4.0 μL of each reaction for the gel.
Add to each reaction 3 μL of loading buffer and 3 μL of DI water or 1X TE Buffer.
Reseal the plate with a new adhesive film.
7. Place the plate back on the thermal cycler and close the lid.
8. Press Resume to continue running the program.
9. Combine the 4.0 μL of each reaction with 3 μL loading buffer and 3 μL DI water
or 1X TE Buffer.
10. Repeat these steps for each Hyb Plate.
11. Load and run the gels.
6.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
EVALUATING THE SECOND QUALITY CONTROL GEL
A good second quality control gel will look like the one shown in Figure 3.54.
The positive control has been
digested and the bands are
identical in size to the sample
bands.
The digested product is now
smaller than the marker band.
Figure 3.54
Good Second Quality Control Gel – Top Half Only
Figure 3.55
Bad Second Quality Control Gel
Three bands are in every well, indicating that incomplete digestion. We recommend
going back to the Assay Plate and running the samples again from Stage 5 – Second
PCR.
chapter 3 | Running the Protocol
83
MARK EXPERIMENTS THAT WILL NOT BE HYBRIDIZED
At this point, you will want to indicate in GTGS any experiments that are not to be
hybridized onto arrays.
To mark experiments that will not be hybridized:
1.
2.
3.
4.
5.
In GTGS, expand the project, Tracking and Hyb Plates folders.
Select the appropriate Label Plate.
Open Tools > Set Well Status.
Right-click the well of each sample you want to fail and select Failed.
Click OK.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Stage 7 – Sample Hybridization
ABOUT THIS STAGE
During this stage, you will add Hyb Cocktail to each sample. The samples are then
placed on a thermal cycler and denatured.
After denaturation, each sample is loaded onto a GeneChip® Universal Tag Array
(array) – one sample per array. The arrays are placed into a hybridization oven that has
been preheated to 39°C. Samples are left to hybridize for 12 to 16 hours.
Each feature on an array contains multiple complementary tags (c-tags.) If you are using
3K arrays, the array would have > 3000 tags.
Label
Tag
Complimentary tag on array
Labelled, cleaved second PCR
product hybridizes to oligos on
Universal Tag Arrays
Figure 3.56
The tag in the assay probe hybridizes to the c-tags on the array surface. If the sample
is that of a homozygote, only one of the four types of allele-specific labelled probes will
hybridize to the c-tags on the feature for the target SNP (for example, probes with the
dATP label).
If the sample is that of a heterozygote, two of the four types of allele-specific labelled
probes hybridize to the c-tags on the feature for the target SNP (for example, probes
with the dATP label and probes with the dCTP label).
LOCATION AND DURATION
• Post-Amp Lab
• Hands-on time: 1 hour
• Time on thermal cycler: 6 minutes
• Hybridization time: 12 to 16 hours
EQUIPMENT AND MATERIALS REQUIRED
The following equipment and materials are required to perform this stage. Quantities
shown are for processing 48 reactions (48 arrays).
chapter 3 | Running the Protocol
Table 3.9
Equipment and Materials Required for Stage 7 – Sample Hybridization
Quantity
Item
1
Aluminum foil, 1 roll
1
Centrifuge, plate
48
Clear film for arrays (enough for one per array; quantities can vary)
48
Universal Tag Arrays (quantities can vary)
2
Hyb Plates containing samples from the previous stage
1
Hybridization oven with trays
1
Ice container, filled with ice
1
Marking pen, fluorescent or water soluble
2
MicroAmp Clear Adhesive Films
1
Microfuge
2
Plate holders (preparation racks)
1
Reagent reservoir, 50 mL
2
Thermal cyclers, 96-well GeneAmp PCR System 9700
KIT COMPONENTS REQUIRED
The following components are required from each GeneChip SNP Kit:
• Hyb Cocktail
THAW REAGENTS
To thaw the reagents:
Wrap the Hyb Cocktail in aluminum foil.
2. Allow to thaw on the bench top.
1.
3.
Place on ice until ready to use.
Hyb Cocktail is light sensitive. Do not expose to light.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
PREPARE THE GENECHIP® UNIVERSAL TAG ARRAYS
To prepare the arrays:
1.
2.
3.
4.
5.
Cover the hybridization oven with aluminum foil to prevent light from entering.
Turn the hybridization oven on now and set to 39°C.
Remove arrays from the 4°C refrigerator.
Unwrap the arrays and place window-side up on a clean bench top.
Cover the window of each array with a piece of clear film.
Figure 3.57
Applying Clear Film to Array Window
6.
Turn over each array so that the window faces down.
Figure 3.58
Array With Sample Numbering
Write a number on each array (Figure 3.58) using a water soluble or fluorescent
marking pen, keeping the arrays in numerical order.
For example, you might number the arrays 1, 2, 3 and so on.
8. Insert a 200 μL pipette tip in the upper right septum of the array.
7.
chapter 3 | Running the Protocol
9.
87
Allow the arrays to warm to room temperature.
To ensure that the data collected during scanning is associated with the correct
sample, number the arrays in a meaningful way. It is critical that you know which
sample is loaded onto each array.
ADD THE HYB COCKTAIL
To add the Hyb Cocktail:
Using a P200 pipette, aliquot 75 μL (150 μL) Hyb Cocktail to each tube in one set
of 12 strip tubes.
2. Using a 12-channel P200 pipette, aliquot 30 μL of Hyb Cocktail to each reaction
on the Hyb Plates (final volume approximately 94 μL).
1.
Final Mix Consists of:
∼ 64 to 68 μL Sample
+ 30 μL Hyb Cocktail
∼ 94–98 μL Total
Mix by pipetting up and down 10 times.
4. Seal Hyb Plates with a new adhesive film.
5. Spin down the plates at 1000 rpm for 30 seconds.
6. If not immediately proceeding to Denature Samples below, Hyb Plates can be stored
at −20°C for up to 2 days.
3.
Hyb Plates can be stored at –20°C for up to 2 days.
We recommend freezing and thawing stored plates ONE TIME ONLY. For a given
set of samples, do not freeze and thaw plates more than once during the protocol.
DENATURE THE SAMPLES
To denature the samples:
1.
Place each Hyb Plate on a thermal cycler, and run the Meg Denature program
(Figure 3.59.)
2.
Immediately after the 6 minute denature:
A.
B.
C.
D.
E.
Remove Hyb Plates from the thermal cycler.
Place on ice.
Cover with aluminum foil to protect from light.
Cool for 2 minutes.
Spin down at 1000 rpm for 30 seconds.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Do not leave on thermal cycler.
Place samples on ice immediately after 6 minutes.
Keep covered until ready to hybridize onto arrays.
Figure 3.59
Meg Denature Thermal Cycler Program
LOAD REACTIONS ONTO ARRAYS
To load reactions onto the arrays:
In GTGS, expand the Project, Tracking and Hyb Plate folders.
2. Right-click the appropriate Hyb Plate and select Hyb on Arrays.
3. Scan the Hyb Plate barcode and press Enter.
The Hyb on Arrays window appears (Figure 3.60 on page 88). Notice the top field
of this window prompts you as to which reaction to load onto an array. When an
array barcode is scanned, the software highlights the next sample to be loaded.
1.
To ensure that the data from the scanner is associated with the correct sample, you
must keep track of which sample is loaded onto which array. Follow the transfer
instructions displayed in the Hyb on Arrays window (Figure 3.60).
Figure 3.60
Hyb on Arrays Window. The software indicates by well designation which reaction is to be loaded
onto an array. After the array barcode is scanned, the software highlights the well of the next
reaction to be loaded.
chapter 3 | Running the Protocol
4.
89
Working 24 arrays at a time:
Set a single channel pipette to 90 μL.
B. Pipette 90 μL of reaction from the plate and well specified in the Hyb on Arrays
window, and load it onto an array through the lower left septum (Figure 3.61).
A.
Figure 3.61
Loading Sample Onto an Array
Remove the pipette tip from the upper right septum.
D. Scan the array barcode.
E. Place the array in a hybridization oven tray.
F. When the tray is full, place and secure the tray in the oven.
C.
Place trays in the oven as soon as the arrays are loaded. Do not leave loaded arrays
on the bench top for more than 5 minutes.
G.
Repeat this process until all of the reactions have been loaded onto arrays and
placed in the hybridization oven.
Ensure that the trays are seated properly in the oven carousel.
6. Set the oven speed to 25 rpm.
7. Leave the arrays in the oven for 12 to 16 hours.
8. In GTGS, click Next, then click Save.
5.
What happens next depends on whether GTGS is configured to communicate with
GCOS, or is configured to write sample files (.arr) for AGCC. See the AGCC and GCOS
Compatibility Modes chapter of the GeneChip® Targeted Genotyping Software User Guide
for more information about GCOS and AGCC modes.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
If you are using AGCC:
Select a new or existing folder within the AGCC data root used by the computer
that will wash and scan your arrays.
GTGS will create sample files (.arr) in this folder, which AGCC needs before arrays
are washed or scanned. AGCC will create data files in this same folder when the
arrays are scanned.
2. Click Save.
3. Check the AGCC Portal to ensure that the information from GTGS has been
transferred and detected (indexed) by AGCC.
Indexing may take up to 10 min after files are added to the AGCC data root.
4. Click Close to close the Hyb on Arrays window.
1.
If AGCC has not indexed the GTGS-created .arr files by the time the arrays are first
washed or scanned, then AGCC will itself create .arr files, which is a problem. When
AGCC creates .cel files, the names will be based on the full array barcode.
If this occurs, GTGS will not be able to load the .cel files. The reason is that GTGS
expects the .cel files to have a name based on the experiment name used in GTGS.
This name is an abbreviation of the full array barcode.
If you accidentally save these files to the wrong location, you can also move your
sample files to the correct location before the arrays are washed and scanned.
Figure 3.62
AGCC data root
chapter 3 | Running the Protocol
91
If you are using GCOS:
After several seconds, a message should appear confirming that the experiments have
been registered with GCOS.
Check GCOS to ensure that the infomration from GTGS has been transferred.
2. Click Close to close the window.
For both AGCC and GCOS, you can click the Arrays folder icon to see a list of arrays
that have been barcode scanned. You will also see information about each array in the
right pane (Figure 3.63).
1.
Figure 3.63
Arrays Icon Expanded and Array Tab
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Stage 8 – Stain and Wash
The instructions for this stage refer to GCOS fluidics station control software. For
Affymetrix GeneChip® Command Console users (AGCC), refer to the AGCC user’s
guide for instructions on controlling the fluidics station.
ABOUT THIS STAGE
During this stage, the arrays are loaded onto a GeneChip® Fluidics Station (fluidics
station). Each array is washed and stained in preparation for scanning.
LOCATION AND DURATION
• Post-Amp Lab
• Hands-on and wash time: approximately 6 hours
Stages 8 and 9 are run concurrently. As arrays come off the fluidics station, you will
load them onto the GeneChip® Scanner 3000 7G 4C and scan them.
EQUIPMENT AND MATERIALS REQUIRED
The following equipment and materials are required to perform this stage. Quantities
shown are for processing 48 arrays. The number of arrays processed can vary.
Table 3.10
Equipment and Materials Required for Stage 8 – Stain and Wash
Quantity
Item
1
Centrifuge, plate
2
Conical tubes, 15 mL
48
Eppendorf tubes, Amber, 2.0 mL (1 tube per array; actual quantities required may
vary)
48
Eppendorf tubes, Natural, 2.0 mL (1 tube per array; actual quantities required
may vary)
48
Universal Tag Arrays from the previous stage (quantity can vary)
1
Ice container, rectangular, filled with ice
KIT COMPONENTS REQUIRED
The following GeneChip SNP Kit components are required for this stage:
• Buffer H
• Stain Cocktail
• Wash Soln A
• Wash Soln B
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93
PRIME THE FLUIDICS STATION
To prime the fluidics station:
1.
Remove Wash Solutions (Solns) A and B from all kits being used.
1.
Turn on the fluidics station and:
Place Wash Solns A and B in the designated positions.
B. Fill the dH2O container.
C. Empty the waste container.
2. Launch GCOS and:
A.
Click the Fluidics button.
B. Navigate to Run → Fluidics → Protocol.
C. Open the Protocol drop-down list and select Prime_450.
A.
Figure 3.64
Selecting the Prime_450 Protocol
Select the All Modules button.
E. Click Run.
F. Follow the instructions on the monitor.
The displays on the fluidics station for each module will read Priming done when
the program is finished.
D.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
PREPARE REAGENTS
To prepare the reagents:
1.
2.
3.
4.
5.
6.
From each kit being used, remove the Buffer H and Stain Cocktail.
Mix the Stain Cocktail by inverting 5 times.
Wrap the Stain Cocktail tube in aluminum foil and place on ice.
Thaw Buffer H on the bench top.
Spin down Buffer H and mix by pipetting up and down.
Keep all reagents on ice until ready to use.
Stain Cocktail is light sensitive. Keep protected from light.
PREPARE THE STORAGE COCKTAIL
To prepare the Storage Cocktail:
In a 15 mL conical tube, add the reagents listed in Table 3.11 in the order shown.
Be sure to dilute Buffer H using Wash Soln B; do not use water.
2. Cap the tube and mix gently by inverting.
1.
Table 3.11
Storage Cocktail
Reagent
1 Array
24 Arrays
(2 extra arrays)
48 Arrays
(4 extra arrays)
Wash Soln B
179 µL
4.65 mL
9.31 mL
Buffer H
11.4 µL
296 µL
593 µL
Total
190 µL
4.95 mL
9.90 mL
ALIQUOT THE STAIN AND STORAGE COCKTAILS
To aliquot the stain and storage cocktails:
1.
For every array, aliquot 190 μL of Stain Cocktail to an amber 2.0 mL Eppendorf
tube (one tube per array).
2.
Keep the tubes on ice and covered to protect from light until ready to use.
Stain Cocktail is light sensitive. To avoid exciting the dyes, keep tubes protected
from light.
3.
For every array, aliquot 190 μL of Storage Cocktail to a clear 2.0 mL Eppendorf
tube (one tube per array.)
4.
Keep the tubes on ice until ready to use.
chapter 3 | Running the Protocol
95
LOAD ARRAYS ONTO THE FLUIDICS STATION
To load arrays onto the fluidics station:
1.
Remove eight arrays from the hybridization oven.
Leave remaining arrays in the hybridization oven until ready to wash.
Place each array on a fluidics station, but do not engage the wash block.
3. In GCOS, click the Fluidics button
.
4. Select the appropriate module.
5. Open the Protocol menu and select TrueTag_Chip_Wash_R7_450.
This step is required when you scan the first array only.
2.
Figure 3.65
Selecting a Wash Protocol on the Fluidics Station
6.
To scan the array barcode:
Highlight the barcode field (or click in the field if this is the first run).
If this is the first run of arrays, the Barcode field is empty. You must still click
in the Barcode field before scanning each of the first four arrays.
B. Scan the array barcode.
C. Wait for the Experiment field to update (Figure 3.66).
A.
You must wait for the Experiment field to update after scanning the array barcode.
If you click Run before the field updates, the scanner will not be able to scan the
array. A message stating that the array was not hybridized is displayed.
After the barcode is scanned, GCOS checks the experiment list and automatically
updates the Experiment field. The experiment name is typically a truncated
version of the array barcode.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
An error message is displayed if GCOS cannot find an experiment that corresponds
with the array barcode.
Updated automatically by
GCOS after the array barcode
is scanned.
Figure 3.66
Experiment Field
Click Run (or press Enter) to proceed to the next module and scan the array
barcode.
8. Repeat until all of the barcodes have been scanned and entered.
9. Follow the prompts on the fluidics station as follows:
7.
When the display reads Load Cartridge, engage the wash blocks.
B. When the display reads Load Stain in 1, Store in 2 (Figure 3.67) :
A.
Load a tube of Stain Cocktail in position 1.
2) Load a tube of Storage Cocktail in position 2.
3) Load an empty tube in position 3.
C. Lower the needles to start the program.
1)
Stain Cocktail in
position 1
Storage Cocktail in
position 2
Figure 3.67
Tube Positions on the Fluidics Station
Empty tube in
position 3
chapter 3 | Running the Protocol
10. If
97
the scanner is not turned on:
Launch GCOS first.
B. Turn the scanner on.
The scanner must warm up for 10 minutes before scanning arrays.
A.
REMOVE ARRAYS FROM THE FLUIDICS STATION
To remove arrays from the fluidics station:
When the Remove Cartridge prompt is displayed, remove the array.
2. Inspect the array for air bubbles. If found:
• For small bubbles, gently swipe the array on the bench top to move the bubble
out of window (Figure 3.68).
• For larger bubbles:
1.
Place the array back on the fluidics station.
2) Re-engage the wash block.
3) Allow the program to finish.
The array is automatically drained and refilled with Storage Cocktail.
Repeat this process as many times as necessary.
1)
The TrueTag_Chip_Wash_R7_450 script is designed to pause automatically so that
you can check the arrays for bubbles, and reinsert the arrays to drain and refill as
many times as necessary.
Figure 3.68
Swiping an Array to Dispel Air Bubbles
Reinspect the array and repeat the previous step if necessary.
4. Re-engage the washblock to restart the TrueTag_Chip_Wash_R7_450 script
and allow it to finish.
5. At the prompt, remove and discard the stain and storage cocktail vials from
positions 1 and 2.
Protocol Done is displayed on the fluidics station when the script is finished. Leave
the empty tube in position 3.
6. Place the arrays face down and cover the septa with Tough Spots.
3.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Do not allow edges of Tough Spots to overlap the large center circle of the array. If
tags overlap, the array may get stuck in the scanner.
Remove the clear film from each array window.
8. Proceed immediately to Stage 9 – Scan Arrays on page 99 and scan the arrays.
7.
PROCESS THE REMAINING ARRAYS
To process the remaining arrays, repeat the steps listed under Load Arrays onto the
Fluidics Station on page 95 and Remove Arrays from the Fluidics Station on page 97.
Load new tubes of Stain and Storage Cocktail for each array. Leave the empty tube
in position 3.
SHUT DOWN THE FLUIDICS STATION
To shut down the fluidics station:
1.
2.
3.
4.
5.
6.
Remove Wash Solns A and B and replace with distilled H2O (dH2O).
Place tubing in dH2O.
Place empty tubes in positions 1, 2 and 3.
Select the All Modules button.
Run the protocol called Shutdown_450 (Figure 3.69).
Turn off the fluidics station.
Figure 3.69
Shutting Down the Fluidics Station
chapter 3 | Running the Protocol
99
Stage 9 – Scan Arrays
The instructions for this stage refer to GCOS scanner control software. For
Affymetrix GeneChip® Command Console users (AGCC), refer to the AGCC user’s
guide for instructions on controlling the scanner.
ABOUT THIS STAGE
During this stage, the arrays are loaded onto the GeneChip® Scanner 3000 7G 4C
(Scanner 3000 7G 4C). Each array is scanned individually, and the data collected is
stored in four files referred to as .cel files. Each .cel file contains the data for one
channel (A, C, G or T). The naming convention for AGCC .cel files created by GTGS
is <abbreviated barcode>_<channel designation>.cel. The naming convention for GCOS
.cel files is <abbreviated barcode><channel designation>.cel. The channel designation is
A, B, C or D.
AGCC Example: @1234567-12345_A.cel
GCOS Example: @1234567-12345A.cel
Data is generated by collecting light from four different wavelengths – one for each
channel. The amount of light emitted by each feature on the array is collected, and the
background is subtracted.
If the sample is from a homozygote, only a single wavelength of light is emitted from
the feature for the target SNP. If the sample is from a heterozygote, two different
wavelengths of light are emitted.
LOCATION AND DURATION
• Post-Amp Lab
• Hands-on and scanning time: approximately 4 to 5 hours
Stages 8 and 9 are run concurrently. As arrays come off the fluidics station, they are
loaded onto the Scanner 3000 7G 4C and scanned.
EQUIPMENT AND MATERIALS REQUIRED
The following equipment and materials are required to perform this stage. Quantities
shown are for processing 48 arrays:
Table 3.12
Equipment and Materials Required for Stage 9 – Scan Arrays
Quantity
1 can
Item
Compressed air
1
GeneChip Scanner 3000 7G 4C
48
Arrays from the previous stage (quantities can vary)
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
PREPARE THE SCANNER
If the scanner is not already on and warmed up:
Launch GCOS first.
2. Turn on the scanner.
3. Allow the scanner to warm up for 10 minutes before scanning arrays.
1.
LOAD THE ARRAYS
To prepare the arrays for scanning:
Inspect the windows for dust, lint or other blemishes.
2. If necessary, clean the array window using compressed air.
3. Load each array the scanner carousel, starting at position 1.
The carousel holds 48 arrays.
1.
SCAN THE ARRAYS
To scan the arrays:
1.
Click the Start button.
The GeneChip Scanner dialog box is displayed.
Figure 3.70
GeneChip Scanner Dialog Box
2.
If the arrays are:
• At room temperature, select First 4 arrays at room temperature.
• If arrays are not at room temperature, deselect the check box. Bringing arrays
to room temperature takes approximately 5 minutes.
If:
• None of the arrays have been scanned, leave Allow rescans deselected.
• One or more of the arrays have already been scanned, select the Allow rescans
check box.
The scanner will identify arrays that have already been scanned. When rescanned,
the associated files will have the suffixes such as _2A, _2B, _2C, and _2D added
to the filename (A for the first rescan, B for the second rescan, and so on.)
4. Click OK.
The scanner processes approximately 11 arrays per hour.
3.
chapter 3 | Running the Protocol 101
To view the scanning process, open View → Scan in Progress.
The scanner must finish autofocusing before you can view the scan.
6. When scanning is finished, do one of the following:
• Leave the arrays in the carousel where they are held at 15°C.
• Remove and store the arrays at 4°C until no longer needed.
5.
We strongly recommend rescanning arrays within 6 to 24 hours of the first scan if
necessary. Data quality may be degraded if arrays are rescanned after 24 hours.
ADDING ARRAYS DURING AN AUTOLOADER RUN
To add arrays while an AutoLoader run is in progress:
1.
Do one of the following:
Click the Add icon.
B. Open Run → Add Chips,
.
The GeneChip Scanner window appears (Figure 3.71).
A.
Figure 3.71
GeneChip Scanner Window
2.
Click Add after Scan.
Do not use the Add Now feature. Use only the Add after Scan feature when working
with Universal Tag Arrays.
Wait until the scanner is finished scanning the current array.
4. When the status on the scanner reads Autoloader Unlock, open the scanner and
add the arrays.
5. Close the scanner.
6. When the following message is displayed, click OK.
3.
Figure 3.72
Resume Prompt
102
7.
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Do one of the following:
• Click the Start icon.
• Click the Resume icon,
.
• Open Run → Resume Scan.
SHUTDOWN THE SCANNER
To shutdown the scanner:
Turn off the scanner by pushing the power button.
chapter 3 | Running the Protocol 103
Rescanning Arrays
GUIDELINES FOR RESCANNING ARRAYS
We strongly recommend that arrays be rescanned if necessary within 6 to 24 hours
of the first scan. Scanning arrays after 24 hours can result in degraded data.
Guidelines for rescanning arrays are as follows.
Table 3.13
Guidelines for Rescanning Arrays
If …
Then …
there is no image when viewing the .dat
file (white screen)
rewash the array with fresh Wash Solution B and
rescan. See Rewash Arrays below.
lint, dust or bubbles are present
clean the array window or rewash the array as
appropriate and rescan. See Rewash Arrays below.
data quality is poor
rescan the array.
the image does not grid properly (.cel file
is missing)
do not rescan the array. Manually apply the grid to
the array image and generate the missing .cel file.
See Manually Regridding Arrays on page 118.
REWASH ARRAYS
To rewash arrays:
Insert a P-200 pipette tip in the upper-right septum of the array.
2. Pipette out the Storage Cocktail from the array.
3. Pipette fresh Wash Solution B through the lower left septum as follows:
1.
Pipette up and down 5 times to fill and drain the array.
B. Repeat the fill and drain with fresh Wash Solution B two more times.
C. Remove Wash Solution B from the array.
4. Slowly refill the array with fresh Storage Cocktail (approximately 100 μL), ensure
there are no air bubbles and rescan immediately.
A.
RESCAN THE ARRAY
To rescan the array:
Load the array into the autoloader.
2. Rescan the array as you would normally.
3. At the prompt Allow Rescans, select Yes.
When the AGCC or GCOS software reads the barcode, it will recognize that this
array has already been scanned. AGCC or GCOS will add suffixes (_2A, _2B, _2C,
and _2D) to the associated filenames to reflect the fact that they are rescans.
1.
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Deleting Projects and Project-Related Information
DELETING A PROJECT
To delete a project, you must delete the project components in the reverse order from
which they were created.
For example, if there are cluster results in the Genotype Results folder, you must:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Delete each cluster genotype that is in the Genotype Results folder.
Delete all of the experiments listed under the Array Data icon in the Experiment
QC Summary.
Experiments may be deleted individually or all at once. If you are using GCOS,
you may also want to go to GCOS Manager and delete all experiments associated
with the project. If you are using AGCC, you may also want to go to the folder
containing .arr and .cel files, and move or delete these as well.
Delete the arrays from the Arrays folder.
Delete each plate in the Hyb Plates folder.
Delete each plate in the Label Plates folder.
Delete each plate in the Assay Plates folder.
Delete each plate in the Anneal Plates folder.
Delete each plate in the Sample Plates folder.
Delete the project icon.
DELETING CLUSTER GENOTYPE RESULTS
To delete cluster genotype results:
Right-click the cluster name.
2. Select Delete.
3. Click Yes.
1.
chapter 3 | Running the Protocol 105
DELETING EXPERIMENTS
Experiments can be deleted only if they are not included in any cluster genotyping
results.
To delete individual experiments:
Click the Array Data icon for your project.
2. Click the Experiment QC Summary tab.
3. Right-click anywhere in the row of the experiment you want to delete and select
Delete.
If you cannot select Delete, the experiment is included in cluster genotype results.
You must delete the cluster genotype results before you can delete the experiment.
4. Click OK.
1.
To delete all experiments at once:
Right-click the Array Data icon for your project.
2. Select Delete All Experiments.
The status bar at the bottom of the the application window will show the deletion
progress. Do not force quit the application during this operation, or all of the
experiments will be restored. Since restoring all experiments takes some time, the
application may be unresponsive until the SQL Server has finished this task.
1.
DELETING ARRAYS
You should delete arrays only after the associated Array Data experiments have been
deleted.
To delete arrays:
Select the arrays folder.
2. In the right pane, right-click the set of arrays to delete and select Delete.
3. Click Yes to confirm deletion of each selected array.
To delete multiple arrays, simply hold down the Enter key to quickly accept each
confirmation message.
1.
DELETING ANNEAL, ASSAY, LABEL AND HYB PLATES
You can delete plates as long as there are no arrays hybridized with samples that
originated from these plates.
To delete these plates, you must delete them in reverse order starting with the Hyb
Plate.
To delete plates:
Select the relevant plates folder.
2. Right-click the set of plates to delete, and select Delete.
3. Click Yes.
To delete multiple plates, simply hold down the Enter key to quickly accept each
confirmation message.
1.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
DELETING SAMPLE PLATES
You can delete a sample plate as long as it is not included in a project. If referenced by
any projects, you must first remove the sample plate from those projects, then delete
the sample plate.
To delete a sample plate:
Delete any projects that reference the sample plate.
See Deleting a Project on page 104.
2. Open the menu in the left pane of the GTGS window and select Samples.
3. Expand the Sample Plates folder.
4. Right-click the sample plate name and select Delete.
You can also select Edit → Delete. If you cannot select Delete, the sample plate
has been used in a project that still exists.
1.
5.
Click Yes.
DELETING SAMPLE INFO
If a sample name in the Sample Info table does not exist in any Sample Plate, you can
remove these samples.
To delete Sample Info:
Open the menu in the left pane of the GTGS window and select Samples.
2. Right-click the Sample Info icon and select Remove Unused Sample Info.
3. Click Yes.
1.
DELETING ASSAY PANELS
You can delete an assay panel as long as it is not included in a project. If referenced by
any projects, you must first delete those projects, then delete the assay panel.
To delete an assay panel:
1.
Delete any projects that reference the assay panel.
See Deleting a Project on page 104.
Open the menu in the left pane of the GTGS window and select Assay Panels.
3. Expand the Assay Panels folder.
4. Right-click the assay panel name and select Delete.
You can also select Edit → Delete.
5. Click Yes.
2.
Chapter
4
Troubleshooting
108
GeneChip® Fluidics Station Troubleshooting
This section covers troubleshooting of issues using GCOS fluidics and scanner
control software. Affymetrix GeneChip Command Console (AGCC) users should
refer to the AGCC User's Guide for how diagnose and recover from fluidics,
scanner, or array-related problems.
FLUID NOT DETECTED
Problem: Fluid not Detected
The GeneChip® Fluidics Station (fluidics station) has detected a problem with either
the Stain Cocktail or the Storage Cocktail. This problem can be caused by:
• No solution in a tube
• Solution not prepared correctly
• Wrong solution in a tube
Messages Displayed
On the Instrument Control Workstation, you will see the following messages (the
word position refers to the fluidics station module in these messages):
Figure 4.1
Fluid Not Detected
Solution
To resolve the error and resume the wash and stain operation:
1.
Determine which cocktail or solution is causing the problem by looking at the Step
field.
2.
If the stain cocktail is causing the problem:
A.
Run the protocol RECOVER1_450 (Figure 4.2).
chapter 4 | Troubleshooting 109
Figure 4.2
Recover1_450 Protocol
Follow the prompts on the fluidics station LCD.
C. When finished, a message similar to the one outlined in red (Figure 4.3)
appears.
B.
Figure 4.3
Fluidics Protocol Complete
At the prompt, remove the array from the wash block.
E. Re-engage the wash block and allow the protocol to finish.
3. If the storage cocktail is causing the problem:
D.
Run the protocol RECOVER2_450.
B. Follow the same steps used for RECOVER1_450 above.
4. Open the Protocol menu and select TrueTag_Chip_Wash_R7_450.
A.
Highlight the barcode field and scan the barcode of the array that failed.
6. Click Resume.
5.
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Figure 4.4
Array Barcode in Experiment Field
7.
At the prompt, click OK.
Figure 4.5
Confirm That Recovery Steps Complete
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Troubleshooting the GeneChip® Scanner 3000 7G 4C
SCANNING STOPPED PREMATURELY
Problem: Scanning Stopped Prematurely
On the Instrument Control Workstation, if the scanner should be actively scanning
but the Scan Status reads Ready, the scanner has lost communication with the
workstation.
The upper blue light on the scanner will be illuminated, indicating that the door is
locked and that the instrument should be scanning.
Solution
To re-establish communication and continue scanning:
1. On the GeneChip® Operating Software (GCOS) tool
2.
3.
4.
5.
6.
7.
8.
bar, click the Stop button.
The upper light on the scanner will turn off indicating the door is unlocked.
At the prompt, click OK.
Open the scanner door, and remove the arrays that have been completely scanned
(scan status reads Scan Complete).
If you do not remove the arrays that have already been scanned, those arrays will
be scanned again once operation resumes.
Reposition the arrays, or add additional arrays so that there is an array in position
1 of the autoloader.
Close the scanner door.
Click the Start button.
At the prompt, select Allow Rescans.
Click OK.
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AUTOFOCUS ERROR MESSAGE
Problem: Autofocus Error Message
If the clear film was left on the array, you will see the message outlined in red below
on the Instrument Control Workstation:
Figure 4.6
Message Displayed in GCOS When Clear Film Left on Array Window
Solution
To fix the problem and continue scanning:
1.
2.
3.
4.
5.
6.
7.
On the GCOS tool bar, click the Stop button.
The upper light on the scanner will turn off indicating the door is unlocked.
Click OK when the prompt appears in GCOS.
Open the scanner door, and remove the array.
Remove the clear film and place the array back in the autoloader.
Close the scanner door.
Click the Start button.
Click OK when the prompt appears.
EXPERIMENT NOT HYBRIDIZED MESSAGE
Problem: Experiment not Hybridized Message
On the scanner you will see the message: The experiment <experiment name> has not been
hybridized (example outlined in red below):
Figure 4.7
Error Message – Experiment Not Hybridized
Cause
This error can occur if you do not allow the Experiments field to update on the fluidics
station when scanning array barcodes. When the field is not allowed to update, the fact
that the array has been stained and washed is not registered with GCOS (even though
the error message says the experiment has not been hybridized).
chapter 4 | Troubleshooting 113
Even though the array barcode is passed to GCOS, the software does not advance the
status of the array to scan. Therefore, when you try to scan the array, GCOS has no
record of the barcode and displays this error message.
Solution
To advance the status of an array to scan:
1.
Click the Workflow Monitor button (or select Workflow Monitor from the
Run menu).
Figure 4.8
Selecting the Workflow Monitor
2.
In the window that appears, open the Assays menu and select Universal.
Figure 4.9
Selecting Universal
A list of arrays may appear.
3. If no list of arrays appears, check you filter settings as follows:
Open Tools → Filters . . .
B. Reset any incorrect fields and click Refresh.
C. If no list of arrays, deselect the Date check boxes and click Refresh.
A.
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D.
Click OK.
Figure 4.10
Selecting Advance to Scan
Locate and right-click the barcode for the array, then select Advance to Scan.
5. Put the array back into the scanner, and scan the array.
4.
chapter 4 | Troubleshooting 115
Troubleshooting and Generating Missing .cel Files
CAUSES OF MISSING .CEL FILES
On occasion, the scanner may generate .dat files but no .cel files for a particular array
or group of arrays. Causes are:
• A software error occurred
When many .cel files are missing, this is typically the cause.
• A gridding error occurred
DETERMINE THAT .CEL FILES ARE MISSING
The first indication that .cel files are missing is when you import experiment data into
Affymetrix GeneChip® Targeted Genotyping Analysis Software (GTGS). The Import
Experiment Data window in GTGS displays:
• The total number of experiments you attempted to process
• The number of experiments actually processed
• The number of experiments that were skipped
To generate missing .cel files, you must first determine the name of the corresponding
.dat files. The two methods we recommend are:
• Viewing the Messages field of the Import Experiment Data window in GTGS
Use this method if less than 5 experiments were skipped due to missing .cel files.
See Locate .dat Filenames in GTGS on page 115.
• Using the MegAllele™ Processing tool in GeneChip® Operating Software (GCOS)
Use this method if more than 5 experiments were skipped due to missing .cel files.
See Locate the .dat File in GCOS on page 119.
LOCATE .DAT FILENAMES IN GTGS
To identify the .dat files that correspond to missing .cel files in GTGS:
In GTGS, look at the Import Experiment Data window.
2. Look for an error similar to the one shown below in the Messages field.
ERROR DAT file exists but CEL file does not exist:
\\DB4YGL51\GCLims\Data\(a)4004602-02693D.cel
Based on the .cel filename, you know that the name of the corresponding .dat file
is (a)4004602-02693D.dat.
Refer to the Affymetrix GeneChip® Targeted Genotyping Analysis Software User Guide for
more information.
1.
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GENERATE MISSING .CEL FILES
Restart the Instrument Control Workstation
If the .cel files are missing due to a software error, restart the Instrument Control
Workstation. Restarting the workstation will force the creation of the missing .cel
files. Once generated, reimport your experiment data into GTGS.
Manually Grid the .dat Files
If the .cel files are missing due to gridding errors, you must manually regrid the .dat
files. Follow the instructions below to locate the .dat filenames and manually apply the
grid.
Locating .dat Filenames in GCOS
To locate .dat files that correspond to missing .cel files in GCOS:
Click the MegAllele Processing button.
Figure 4.11
MegAllele Processing
The Status window displays the messages “Searching for unprocessed MegAllele
files…” and “Reading .dat file headers…” as shown in Figure 4.12.
Messages displayed by
GCOS while searching
for missing .cel files.
Figure 4.12
Missing CEL File Error Messages
Next, a table listing the .dat files is displayed (Figure 4.13).
Figure 4.13
List of DAT Files
chapter 4 | Troubleshooting 117
The software searches the entire list of .dat files in the system; therefore, this procedure
can take some time. The search is complete when the Status window displays the
message “<number> unprocessed MegAllele files found (example shown in Figure
4.14).
Figure 4.14
Unprocessed MegAllele Files Message
Opening .dat Files to Fix Gridding Errors
To open .dat files:
1.
Do one of the following:
• Select the filename and click the Open button.
• Double-click the filename.
• Click the Select All icon; then click the Open button.
Figure 4.15
Opening a DAT File
When you see the message Cell grid is not aligned in C:\<path to .dat file>, click OK.
The DAT file opens and is displayed with an un-aligned grid.
3. Manually align the grid and click Calculate CEL Intensity.
The .cel file is generated automatically.
For instructions on manually aligning a grid, see Manually Aligning a Grid on
page 120.
2.
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Manually Regridding Arrays
WHAT YOU WILL SEE
When scanning arrays, the gridding step can sometimes fail. When this occurs, a .cel
is not generated from the corresponding .dat file. You can determine that a .cel file has
not been generated in various ways:
• When importing .cel files to GTGS, an error message is displayed stating that a
particular .cel file could not be found.
• In GCOS, the following gridding error message is displayed in the Scan Status
column of the lower left window:
Failed to align grid. Error returned is: Failed to align the grid in Checkerboard Grid Align.
Corrective action to manually align the grid.
Full text of message displayed in Scan Status window.
Figure 4.16
Gridding Error Message
• In GCOS in the lower right window, no information is displayed for .cel files that
were not generated. In the example below, no .cel file was generated for the A
channel for experiment (a)4003192-25600. Only channels B, C and D have .cel files
(Figure 4.17).
Figure 4.17
chapter 4 | Troubleshooting 119
Figure 4.18 shows a .dat file that did not grid properly. You can see how the grid is
not aligned with the edges of the array image.
Corner of grid
Corner of array image
Figure 4.18
Example of .dat File That Did Not Grid Properly
LOCATE THE .DAT FILE IN GCOS
To locate the .dat file on the Instrument Control Workstation:
On the workstation, expand the Image Data folder.
2. Double-click the .dat file that corresponds to the missing .cel file.
1.
Figure 4.19
Image Data Folder and DAT Files
3.
Click OK.
The array image is displayed.
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MANUALLY ALIGNING A GRID
To manually align a grid:
1.
If a grid is not already displayed over the array image, click the Grid button.
The Grid button is located in the lower right corner of the array image window.
Grid button
Figure 4.20
Grid Button
The misaligned grid is applied over the array image (Figure 4.21).
Click and drag each corner of the grid
to each corner of the array image
Figure 4.21
Misaligned Grid
2.
Perform a preliminary alignment as follows:
Click and hold the left mouse button on a corner of the grid.
B. Drag and align the grid over the array image.
C. Repeat this procedure for each corner of the grid.
3. Perform a fine alignment as follows:
A.
A.
Jump to a corner of the grid using one of these methods:
- Press F5, F6, F7 or F8 on the keyboard
- Click the corners icon to expand it; then click one of the corners (Figure 4.22).
chapter 4 | Troubleshooting 121
Corners icon
Click one of the boxes to jump
to the respective corner
Figure 4.22
Corners Icon
B.
Click and drag the corner of the grid over the array image as shown in the
following illustration.
Grid before fine alignment
Grid after fine alignment
Figure 4.23
Grids Before and After Fine Alignment
C.
Click Recalculate Cel Intensity.
GENERATE A .CEL FILE
To generate a .cel file:
Place the mouse over the image, right-click and choose Recalculate Cell
Intensity.
2. Update the .dat and .cel files in the Image Data and Cell Intensities folders as
follows:
1.
Open Tools → Filters → Refresh.
B. Click OK.
A.
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Identifying Skipped Experiments
HOW TO IDENTIFY SKIPPED EXPERIMENTS
Two methods are available for identifying skipped experiments. You can:
• View the Import Experiment Data window as .cel files are being imported and
processed.
• View the Experiment QC Summary table.
VIEWING THE IMPORT EXPERIMENT DATA WINDOW
Experiment data is imported to GTGS and processed via the Import Experiment Data
window. Once the software is finished reading and processing .cel file data, look at the
Processed/Skipped/Total field to see if any experiments were skipped. In the
example shown in Figure 4.24, one experiment was skipped.
Figure 4.24
Identifying Skipped Experiments in the Import Experiment Data Window
For instructions on troubleshooting skipped experiments via the Import Experiment
Data window, refer to the Affymetrix GeneChip® Targeted Genotyping Analysis Software
User Guide.
chapter 4 | Troubleshooting 123
VIEWING THE EXPERIMENT QC SUMMARY WINDOW
If the Import Experiment Data window has already been closed, you can identify
skipped experiments by looking at the Experiment QC Summary table. The columns
that hold values for the QC metrics (QC Call Rate, QC Half Rate, and so on) will be
empty as shown in Figure 4.25. Refer to the Affymetrix GeneChip ® Targeted Genotyping
Analysis Software User Guide for information on QC metrics.
Skipped experiments
Figure 4.25
Identifying Skipped Experiments in the Experiment QC Summary Table
To troubleshoot skipped experiments found in the Experiment QC Summary table:
Select the Array Data icon for your project.
2. In the Experiment QC Summary table, look for rows where the QC Metric
columns are empty (Figure 4.25).
3. Determine why the data was not imported as follows:
1.
Right-click the Array Data icon for the project and select Import
Experiment Data.
The Import Experiment Data window appears.
B. Look at the Ready to Process field for experiments that were skipped.
The names of the experiments you identified in the Experiment QC Summary
table should appear in the Ready to Process list.
A.
These experiments
correspond to the skipped
experiments in the
Experiment QC Summary
table
Figure 4.26
Identifying Skipped Experiments
C.
Look at the Messages field and do one of the following:
- If the Messages field is empty, click Process. The software will run through
the list of experiments again and will display error messages related to the
skipped experiments.
- If the Messages field is not empty, scroll through the messages for the ones
related to the skipped experiments.
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Troubleshooting Failed Experiments
For more information on GTGS and reviewing data quality, refer to the Affymetrix
GeneChip® Targeted Genotyping Analysis Software User Guide.
ABOUT DATA PROCESSING
Data processing occurs as .cel files are imported into GTGS. During processing,
GTGS calculates and compares the results of each experiment against the following set
of quality control metrics ():
• QC Call Rate
• QC Half Rate
• Signal to Background (median ratio)
• Controls Coefficient of Variation (Controls CV %)
Based on this analysis, GTGS automatically assigns the status of Pass or Fail to each
experiment. The threshold for each of these metrics must be met for an experiment to
be assigned the status Pass. If any metric is not met, the experiment is assigned the
status Fail.
Table 4.1
Quality Control Metrics
Quality Control Metric
Description
Threshold
QC Call Rate %
Estimated percentage of assays clearly
genotyped.
Must be ≥ 80%
QC Half Rate %
Estimated percentage of assays
marginally genotyped.
Must be ≤ 10%
Signal to Background
Median ratio of assay allele/non-allele
channel signals (median signal to
background ratio)
Controls CV%
(Controls coefficient of variation)
Percent coefficient of variation of
control feature signals.
Must be ≥ 20
Must be ≤ 30%
IDENTIFYING FAILED EXPERIMENTS
To identify failed experiment in GTGS:
Click the Array Data icon for your project.
2. In the Experiment QC Summary table, sort on the Hyb Date column so that the
latest imports are displayed first.
As experiment data is imported into GTGS, it is appended to the bottom of the
list.
1.
3.
Look at the Auto column for experiments with the status Fail.
chapter 4 | Troubleshooting 125
TOOLS AVAILABLE FOR REVIEWING YOUR DATA
The following tools are available in GTGS for reviewing your data prior to cluster
genotyping:
• Experiment QC Summary
• Experiment Metrics Chart
• Channel Metrics Chart
• Experiment Details including:
- Summary
- Channel Details
- Array View
For more information on these tools and how to work with them, refer to the
Affymetrix GeneChip® Targeted Genotyping Analysis Software: User Guide.
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TROUBLESHOOTING FAILED EXPERIMENTS
To troubleshoot failed experiments:
Look at the QC Call Rate for the failed experiments.
2. Click the Experiment Metrics Chart tab.
3. Select these display options:
Y Axis: Call Rates %
X Axis: Experiment Name
1.
Table 4.2
Troubleshooting Failed Experiments
If …
Then …
Suggested Actions
Many experiments have low
call rates
compare sample call
rates to control call rates
by toggling the Controls
check box on and off.
If Kit Control data Auto status is Pass, but many experiments were
automatically failed, the quality of your sample DNA is suspect.
See About Your DNA Samples on page 2 for sample criteria.
If both samples and controls failed, an error was made while
performing the protocol. Rerun the samples.
Individual experiments seem
to have randomly failed
Open the Array View
and look at the control
features.
• If Kit Control data Auto status is Pass, but many experiments
were automatically failed, the quality of your sample DNA is
suspect. See About Your DNA Samples on page 2 for sample
criteria.
• If both the border control features and the text are clearly seen
(Figure 4.27), then the array hybridization and stain/wash steps
were correctly performed. Possible cause: assay performed
incorrectly prior to Stage 8 – Preparation for Sample
Hybridization.
- Look at second stage gel. Was there product of correct size in
all 4 lanes? If yes, then at some point between target digest
and hyb on arrays something went wrong. Solution: rerun
samples from stage 5 on.
- If the second stage gel looks bad, go to first stage gel. If first
stage gel looks good, then rerun samples from stage 5 on.
- If first stage gel bad, then rerun the samples from stage 1 on.
If control features are not
uniform across the array
• Look at the Experiment QC Summary and sort on Controls CV %.
• there may be a
If the value is over 30%, the experiment is failed automatically.
hybridization problem
• the array may be
defective
• Look at the .cel file borders in the Array view.
Possible causes:
• Hybridization oven stopped
• Temperature in Hybridization oven too high or too low
• Fluidics station error such as wrong buffers used. Look at log for
errors.
• Excess stain.
Solution:
• Rewash and rescan the array. See Rescanning Arrays on
page 103 for instructions.
• Rerun the samples.
• Arrays may be defective.
chapter 4 | Troubleshooting 127
Figure 4.27
Controls Features Present; No Signal for Assay Features
Figure 4.28
Control Features Not Uniform
To understand sample data quality more thoroughly, look at all of the X and Y axis
display options in both the Experiment Metrics Chart and the Channel Metrics Chart.
For example, in the Channel Metrics Chart the typical signal strength (signal-to-noise)
of each nucleotide is displayed.
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In the Experiment Metrics Chart, signal-to-background correlates strongly with QC
Call Rate. In this context, background is signal in the non-allele channels. When the
QC Call Rate is high, signal-to-background is a more sensitive metric for detecting
trends in data quality. Low signal-to-background can indicate one of the following:
• Low sample concentration
• Poor amplification
• Sample contamination
High QC half rates can indicate:
• Sample contamination
• Reactions with multiple DNA samples mixed together
Chapter
5
Affymetrix GeneChip® Targeted Genotyping
Analysis Software
130
About Affymetrix GeneChip® Targeted Genotyping
Analysis Software
SOFTWARE OVERVIEW
Affymetrix GeneChip® Targeted Genotyping Analysis Software (GTGS) provides a
full set of tools to help you generate and manage the highest quality genotypes using
the MIP Assay Protocol. This software supports networked client-server environments
with multiple users and large experimental data sets.
System management functionality includes:
• User management
• Protocol management
• Array definition management
Project management functionality includes:
• Experiment design
• Sample tracking through the genotyping process.
- See Project Management on page 141 for an overview.
- Instructions for using the software throughout the various steps of the protocol is
presented in Chapter 3, Running the Protocol.
Data analysis functionality includes:
• Generating genotypes from the data collected by the GeneChip® Scanner 3000 7G
4C (Scanner 3000 7G 4C).
• Managing and overseeing the quality of the array data stored in the Affymetrix
GeneChip® Scanner 3000 Targeted Genotyping System database.
Refer to the Affymetrix GeneChip® Targeted Genotyping Analysis Software User Guide for
further information and instructions.
System Management
USER MANAGEMENT
User management functions include:
• Adding users
• Activating and deactivating users
• Deleting users
A user cannot be deleted if the user name is associated with data.
• Modifying user properties
• Changing user names without logging out
chapter 5 | Affymetrix GeneChip® Targeted Genotyping Analysis Software 131
Adding Users
You must be logged in as admin to add users.
To add a new user:
In the left pane, open the menu and select Users.
2. Do one of the following:
• Open File → New User.
• Right-click the Users folder and select New User (Figure 5.1).
1.
Figure 5.1
Users window
The User window appears.
3. In the Name field, enter a user name.
Figure 5.2
Enter User Name and Description
Optional: In the Description field, enter a description for the user.
5. Click Save.
When created, the user name and description are added to the table in the right
pane of the Users window as shown in Figure 5.3.
4.
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Figure 5.3
New User Displayed in Users Window
Deleting Users
A user cannot be deleted if the user name is associated with any experiments or data.
To prohibit a user whose name has been associated with data from logging on to
GTGS, you must deactivate them. See Deactivating Users on page 133.
To delete a user:
Open the menu and select Users.
2. Select the user name.
3. Do one of the following:
• Open Edit → Delete.
• Right-click the user name and select Delete (Figure 5.4).
4. At the confirmation prompt, click Yes.
1.
Figure 5.4
Deleting a User
chapter 5 | Affymetrix GeneChip® Targeted Genotyping Analysis Software 133
Changing Users
While using the software, you can switch from one user name to another without
logging out by using the Set User command.
To change users:
Open the menu and select Users.
2. Open Tools → Set User …
3. Open the User Name menu and select a user.
4. Click OK.
1.
Deactivating Users
Users can be deactivated to prohibit them from logging in to GTGS. This
functionality is useful when a user has experiments or data associated with their name
and therefore cannot be deleted. You must be logged in as admin to deactivate users.
To deactivate a user:
Log on to GTGS or Set User as admin.
2. Open the menu and select Users.
3. Do one of the following:
• Select the user name and open Tools → Deactivate.
• Right-click the user name and select Deactivate.
As shown below, notice there is now a Y in the InActive column for that particular
user.
1.
Figure 5.5
Deactivating a User
Activating Users
You must be logged in as admin to activate users.
To activate a user that has been deactivated:
1.
Log on to GTGS or Set User as admin.
Open the menu and select Users.
3. Do one of the follow:
• Select the user name and open Tools → Activate.
• Right-click the user name and select Activate.
Notice that the Y in the InActive column has been removed. The user status is now
active.
2.
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Modifying User Properties
If you are logged in as admin, you can change the user name and description. If you
are logged in as the user, you can modify the user description only.
To modify user properties:
Do one of the following:
• Select the user name and open Edit → Properties.
• Right-click the user name and select Properties.
2. Edit the user properties as permitted.
3. Click Save.
1.
Exporting a List of Users
You can export the display in the right pane of the Users window as a text file.
To export a list of users:
Open File → Export Table.
2. Browse to the location where you want the file saved.
3. In the File name field, enter a name for the text file.
4. Click Save.
1.
chapter 5 | Affymetrix GeneChip® Targeted Genotyping Analysis Software 135
PROTOCOL MANAGEMENT
A protocol designation must be entered into GTGS before samples can be processed
using the MIP Assay Protocol. When running the protocol in the lab, users are asked
to enter a protocol barcode. Entering the protocol barcode enables future users to see
which procedure was followed in the lab when processing a particular set of samples.
Protocol management functions include:
• Adding protocols
• Deleting protocols (if the protocol has not been used)
• Modifying protocol properties
Adding Protocols
To add a protocol:
Open the menu and select Protocols.
2. Select the Protocols folder and do one of the following:
• Open File → New Protocol.
• Right-click the Protocols folder and select New Protocol (Figure 5.6).
1.
Figure 5.6
Add a New Protocol
3.
In the Protocol Creation Dialog window:
In the Name field, enter a name for the protocol.
B. Optional: In the Description field, enter a description for the protocol.
C. Click the Barcode tab.
D. In the Enter Barcode field, scan or type a barcode for the protocol.
Barcodes must contain the prefix PCL_. A barcode for the MIP Assay Protocol
is provided on the quick reference card, MIP Assay Protocol Barcodes. Barcode
designations entered here are associated with experiments run in the laboratory
using the same protocol.
A.
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Figure 5.7
Entering New Protocol Information
4.
Click Save.
The protocol properties are displayed in the right pane of the Protocols window.
Figure 5.8
New Protocol Displayed in Protocols Window
Deleting Protocols
You can delete a protocol only if it is not associated with any experiments or data.
To delete a protocol:
Open the menu and select Protocols.
2. Do one of the following:
• Select the protocol name and open Edit → Delete.
• Right-click the protocol name and select Delete.
3. At the Confirm Deletion prompt, click Yes.
1.
Modifying Protocol Properties
You can modify the name and description of a protocol. You cannot modify the
protocol barcode.
To modify protocol properties:
Open the menu and select Protocols.
2. Select the protocol name and do one of the following:
• Open Edit → Properties.
• Right-click the protocol name and select Properties.
3. Modify the protocol name or description.
4. Click Save.
1.
chapter 5 | Affymetrix GeneChip® Targeted Genotyping Analysis Software 137
Exporting a List of Protocols
You can export the display in the right pane of the Protocols window as a text file.
To export a list of protocols:
1.
2.
3.
4.
5.
Open the menu and select Protocols.
Open File → Export Table.
Browse to the location where you want the file saved.
In the File name field, enter a name for the text file.
Click Save.
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ARRAY DEFINITION MANAGEMENT
An array definition file identifies each feature on an array. Based on this file, the
software is able to distinguish control features from genotyping features. You cannot
analyze data on an array if the corresponding array definition file is not present in the
software.
Array definitions are installed during GTGS installation and configuration. If your
assay panel uses an array type not yet defined in the software, you will need to import
it before proceeding with the MIP Assay Protocol.
Array definition management functions include:
• Adding array definitions
• Deleting array definitions
• Modifying array definition properties
Adding Array Definitions
1.
2.
3.
4.
5.
Load the array definition CD-ROM onto the appropriate computer.
In GTGS, open the menu and select Array Definitions.
Do one of the following:
• Open File → Import Array Definition.
• Right-click the Array Definitions folder and select Import Array Definition.
In the Array Definition File window, locate the filename and select it.
Click Open, then click OK.
Notice the array definition is now displayed (Figure 5.9).
Figure 5.9
Adding an Array Definition
Deleting Array Definitions
An array definition cannot be deleted once an assay panel that references the definition
is imported.
To delete an array definition:
In the software, open the menu and select Array Definitions.
2. Do one of the following:
• Select an array name and open Edit → Delete.
• Right-click the array name and select Delete.
3. Click Yes.
1.
chapter 5 | Affymetrix GeneChip® Targeted Genotyping Analysis Software 139
Modifying Array Definition Properties
You can modify the description of an array definition.
To modify the description of an array definition:
1.
2.
3.
4.
5.
In the software, open the menu and select Array Definitions.
Select an array name.
Do one of the following:
• Open Edit → Properties.
• Right-click the array name and select Properties.
Enter or modify the description.
Click Save.
Displaying Array Definition Details and Features
You can display general details about a particular array definition such as the number
of features on that type of array. You can also display details about the features of a
particular array type such as the Feature ID and Tag ID.
To display array definition details:
Open the menu and select Array Definitions.
2. Select the Array Definitions folder.
3. Click the Arrays Definition Details tab.
Details are displayed in the right pane.
1.
Figure 5.10
Displaying Array Definition Details
To display feature information for a particular type of array:
Open the menu and select Array Definitions.
2. Select an array definition name.
As shown below, information for each feature on the array is displayed in the right
pane of the window.
1.
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
X and Y coordinates
of feature on the array
Universal Tag Array
Numerical value for
each feature
Identifier for each
tag on the array
Figure 5.11
Feature Information for a Particular Type of Array
Exporting a List of Array Definitions
You can export the information in the right pane of the Array Definitions window as
a text file.
To export a list of array definitions:
1.
2.
3.
4.
5.
6.
Open the menu and select Array Definitions.
Select the Array Definitions folder.
Open File → Export Table.
Browse to the location where you want the file saved.
In the File name field, enter a name for the text file.
Click Save.
Exporting A List of Array Features
You can export a list of the features for a particular array as a text file.
To export a list of array features:
Open the menu and select Array Definitions.
2. Select the name of an array.
3. Open File → Export Table.
1.
Browse to the location where you want the file saved.
5. In the File name field, enter a name for the text file.
4.
6.
Click Save.
chapter 5 | Affymetrix GeneChip® Targeted Genotyping Analysis Software 141
Project Management
ABOUT PROJECTS
In GTGS, sample tracking information is organized in folders called projects. Each
project contains information about all of the samples that have been tested (or will be
tested) using a specific assay panel.
Each project you create will ultimately contain the following information:
• Sample Plates
Contains information on the sample plates to be used for a particular project.
Information includes the sample plate barcode, sample names and well contents.
• Anneal Plates
Contains information used to determine which samples are to be transferred:
- First, from a specific Sample Plate to a specific Anneal Plate.
- Then from the Anneal Plate to a specific Assay Plate.
Information includes Sample, Anneal and Assay Plate barcodes, and well or row
designations on all plates.
• Assay Plates
Contains information used to determine which samples are to be transferred:
- First from a specific Anneal Plate to a specific Assay Plate.
- Then from the Assay Plate to a specific Label Plate.
Information includes Anneal, Assay, and Label Plate barcodes, and well or row
designations on all plates.
• Label Plates
Contains information used to determine which samples are to be transferred:
- First from a specific Assay Plate to a specific Label Plate.
- Then from the Label Plate to a specific Hyb Plate.
Information includes Assay, Label, and Hyb Plate barcodes, and well or row
designations on all plates.
• Hyb Plates
Contains information used to determine which samples are to be transferred:
- First from a specific Label Plate to a particular Hyb Plate.
- Then from the Hyb Plate to a specific array.
Information includes Label Plate, Hyb Plate and array barcodes, and well or row
designations on all plates.
• Arrays
Contains information on the arrays used for a particular project including the array
barcode and the date the barcode was scanned.
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ARRAY DEFINITIONS AND ASSAY PANELS
An array definition for the type of array referenced by an assay panel must be imported
into GTGS before you can import the panel information.
DELETE FUNCTIONALITY
Project components must be deleted in the reverse order of how they were added.
You must be logged in as admin to delete arrays. See Deleting Projects and Project-Related
Information Deleting Projects and Project-Related Information on page 104.
EXPORT FUNCTIONALITY
You can export any project information displayed in the right pane as text file. An
example of what would be exported from the Sample Plates window is shown in
Figure 5.12.
The information that would be exported is outlined in red.
Figure 5.12
Exporting Sample Plate Information
To export project information:
Navigate to the window with the desired information.
2. Open File → Export Table.
3. Navigate to the location where you want the text file saved.
1.
In the File Name field, enter a name for the text file.
You do not need to add the extension .txt.
5. Click Open.
4.
Appendix
A
Vendor Contact Information
144
Vendor Contact Information
Customers outside the United States should check each vendor’s website for
additional contact information.
Table A.1
Vendor Contact Information
Company
Address
Telephone
Affymetrix
www.affymetrix.com
3380 Central Expressway
Santa Clara, California 95051
U.S. 1-888-362-2447
U.K. +44 (0)1628 552550
Japan +81-3-5730-8222
Applied Biosystems
www.appliedbiosystems.com
850 Lincoln Centre
Foster City, California 94404
1-800-327-3002
1-650-638-5800
Axygen Scientific, Inc.
www.axygen.com
33210 Central Ave.
Union City, California 94587
1-800-4-AXYGEN
1-510-494-8900
Distributors for Barcoded 96-Well PCR Thermal Cycler Plates and Barcode Labels (Anneal, Assay, Label, Hyb)
For distributors in countries not listed, refer to:www.axygen.com/Distributors
U.S.A.
E & K Scientific, Inc.
3575 Thomas Road
Santa Clara, California 95054
www.eandkscientific.com
Australia
Radiometer Pacific P/L PO Box 47
Nunawading
Vic 3131
oceania1.radiometer.com
Canada
Ultident Scientific
4850 Chemin Bois Franc Suite 100
St. Laurent, Quebec H4R 2G7
www.ultident.com
Tel: 1-800-934-8114 or 1-408-378-2013
Fax: 1-408-378-2611
Tel: [61] 39-7063-655
Fax: [61] 39-7063-755
Tel: 514-335-3433
Fax: 514-335-0992
Germany
Tel: [49] 02551-864310
G.Kisker GbR
Produkte f.d. Biotechnologie Postfach 1329 Fax: [49] 02551-864312
48543 Steinfurt
www. kisker-biotech.com
Japan
Funakoshi Co Ltd.
9-7. Hongo 2-Chome
Bunkyo-Ku
Tokyo 113-0033
www.funakoshi.co.jp
Tel: [81] 3-5259-5901
Fax: [91] 3-5259-1005
appendix A | Vendor Contact Information 145
Table A.1
Vendor Contact Information
Company
Address
Singapore
Bio Laboratories
10 Ubi Crescent. Lobby A #06-02. Ubi
Techpark 408564
www.biolab.com.sg
United Kingdom
Thistle Scientific Ltd.
DFDS House
Goldie Road Uddingston.
Glasgow G71 6NZ
Telephone
Tel: [65] 6846-7577
Fax: [65] 6846-7477
Tel: [44] 169-833-8844
Fax: [44] 169-833-8880
Bio-Rad Laboratories
www.biorad.com
2000 Alfred Nobel Dr.
Hercules, California 94547
Life Science Research Group
1-800-424-6723
1-510-741-1000
BioSmith Biotech
www.biosmith.com
3649 Conrad Ave.
San Diego, California 92117
1-800-929-7894
1-858-270-8389
Clontech, a TAKARA BIO
Company
www.clontech.com/clontech
1290 Terra Bella Avenue
Mountain View, CA 94043
USA
1-800-662-2566
1-650-919-7300
Coriell Cell Repositories
http://locus.umdnj.edu/ccr/
403 Haddon Avenue
Camden, NJ 08103
Tel: 1-800-752-3805
Fax: 1-856-757-9737
Eppendorf
U.S. only: www.eppendorfna.com
Also available through distributors
One Cantiague Road
P.O. Box 1019
Westbury, NY 11590-0207
1-800-645-3050
Gilson
www.pipetman.com
3000 W. Beltline Hwy.
P.O. Box 620027
Middleton, WI 53562-0027
1-800-GILSON1 or 1-608-836-1551
Invitrogen Life Technologies
www.invitrogen.com
1600 Faraday Ave.
P.O. Box 6482
Carlsbad, California 92008
1-760-603-7200
Rainin Instrument, LLC
www.rainin.com
7500 Edgewater Dr.
P.O. Box 2160
Oakland, California 94621
1-800-472-4646
1-510-564-1600
Stratagene
www.stratagene.com
11011 North Torrey Pines Rd.
La Jolla, California 92037
1-858-535-5400
USA Scientific, Inc
www.usascientific.com
P.O. Box 3565
Ocala, Florida 34478
U.S.: 1-800-522-8477
International: 1-352-237-6288
VWR International
www.vwr.com
1310 Goshen Pkwy.
West Chester, Pennsylvania 19380
1-800-932-5000
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Appendix
B
Thermal Cycler Programs
148
Thermal Cycler Programs
The MIP Assay Protocol has been optimized for use with the 96-well GeneAmp® PCR
System 9700 Thermal Cyclers (silver block) manufactured by Applied Biosystems.
Equivalence tests have been run on the other models listed in Chapter 2, Equipment,
Supplies, Consumables.
Five thermal cycling programs are used throughout the protocol. This appendix
briefly describes each of these programs. It also includes step-by-step instructions for
programming the Meg 22 (20) cycle programs.
PRE-AMP LAB THERMAL CYCLER PROGRAMS
Only the GeneAmp PCR System 9700 Thermal Cyclers are approved for use in the
Pre-Amp Lab. These thermal cyclers must be set up to run the following programs:
• Meg Anneal
• Meg 22 (20) cycle programs
- Meg 3-5-10k
- Meg 20k
POST-AMP LAB THERMAL CYCLER PROGRAMS
The thermal cyclers listed under Thermal Cyclers on page 13 can be used in the PostAmp Lab. These instruments must be set up to run the following programs:
• Meg Hypcr programs
- Meg Hypcr 3-5k
- Meg Hypcr 10-20k
• Meg Hydigest-a
• Meg Denature
SETTING THE RAMP SPEED AND VOLUME FOR EACH PROGRAM
The first time you run each of these programs, you must change the default ramp
speed and volume settings.
Ramp Speeds
Use the following ramp speeds for the thermal cyclers validated for use with the
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System.
• GeneAmp PCR System 9700 Thermal Cycler with a gold or silver block: Max
• GeneAmp PCR System 9700 Thermal Cycler with an aluminum block: Std
• GeneAmp PCR System 9700 Thermal Cycler with a dual block: 96D
• DNA Engine® Peltier and Dyad: Gradient
appendix B | Thermal Cycler Programs 149
Setting Ramps Speeds and Volumes
The following instructions are for programming a GeneAmp PCR System 9700
thermal cycler with a gold or silver block.
To set the ramp speed and volume for each program:
1.
2.
3.
4.
5.
6.
Press Run (F1).
Use the arrow pad to select the program.
Press Start (F1).
Press the down arrow to move to the ramp speed.
Press Max (F3).
Max is the ramp speed to use for GeneAmp PCR System 9700 thermal cyclers with
a gold or silver block. For other cycler ramp speeds, see Ramp Speeds above.
Press the up arrow to move to the reaction volume and enter the volume
appropriate volume:
• Meg Anneal: 45
• Meg 3-5-10k and Meg 20k: 67
• Meg Hypcr 3-5k and Meg Hypcr 10-20k: 35
• Meg Hydigest-a: 75
• Meg Denature: 90
7. Press Start (F1) to start the program.
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Meg Anneal Thermal Cycler Program
ABOUT THE MEG ANNEAL PROGRAM
The Meg Anneal program consists of three holds and no cycles.
Ramp speed and volume:
• Ramp speed
- GeneAmp PCR System 9700 with a gold or silver block = Max
- GeneAmp PCR System 9700 with an aluminum block = Std
• Volume: 45 μL
The ramp speed and volume must be set the first time you use the program. See
Setting the Ramp Speed and Volume for Each Program on page 148.
Table B.1
Stages of the Meg Anneal Thermal Cycler Program
Stage
Temperature
Time
Enzyme A
20°C
4 minutes
Denature
95°C
5 minutes
Anneal
58°C
Infinity
Figure B.1
Meg Anneal Thermal Cycler Program
appendix B | Thermal Cycler Programs 151
Figure B.2
Thermal Cycler Display for the Meg Anneal Program
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Meg 22 (20) Cycle Thermal Cycler Programs
ABOUT THESE PROGRAMS
Meg 3-5-10k and Meg 20k are two variations of the same thermal cycling program.
The difference between these programs is the number of cycles used for the denature,
anneal and extend stages at the end of the program (Table B.2). The version that you
will use is determined by the assay panel size. The only difference between these
programs is the number of amplification cycles performed.
• For 3K, 5K and 10K assay panels: use Meg 3-5-10k (22 cycles)
• For 20K assay panels: use Meg 20k (20 cycles)
Ramp speed and volume for both programs:
• Ramp speed
- GeneAmp PCR System 9700 with a gold or silver block = Max
- GeneAmp PCR System 9700 with an aluminum block = Std
• Volume: 67 μL
The ramp speed and volume must be set the first time you use the program. See
Setting the Ramp Speed and Volume for Each Program on page 148.
Table B.2
Stages of the Meg 22 (20) Cycle Thermal Cycler Programs
Stage
Temperature
Time
Gap Fill/Split
58°C
2 minutes
dNTP Mix/Anneal
58°C
10 minutes
Ligate
58°C
10 minutes
Exo
37°C
15 minutes
Denature
95°C
5 minutes
Cleavage Mix
37°C
10 minutes
Amp Mix
60°C
2 minutes
Denature
95°C
10 minutes
Denature
95°C
20 seconds
Anneal
64°C
45 seconds
Extend
72°C
10 seconds
72°C
10 seconds
4°C
Infinity
Finish
Cycles
3K/5K/10K: 22 cycles
20K: 20 cycles
appendix B | Thermal Cycler Programs 153
Figure B.3
Meg 22 (20) Cycle Thermal Cycler Programs
TO SET UP THE MEG 22 (20) CYCLE PROGRAMS
The GeneAmp PCR 9700 thermal cyclers accept six holds only. Because these
programs include 10 holds, the instrument must be programmed using an
unconventional method. Essentially the thermal cycler is programmed to accept two
high-level hold programs: one with 6 holds; the other with 2 holds.
To set up the Meg 22 (20) cycle programs, you will:
• Insert an additional cycle program
• Change one of the cycles to a hold and create the first 8 holds
• Set the temperatures and times for the first 8 holds
• Set the temperatures and times for the amplification cycle
• Set the temperatures and times for the last 2 holds
• Store the program
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Insert Addition Cycle Program
To insert an additional cycle program:
1.
Turn on the thermal cycler.
2.
Press F2 to select Create.
Figure B.4
The generic program appears. The 1 of 1 Hld is highlighted.
Figure B.5
3.
Press the down arrow to highlight 94.0 and display different options above the
function keys.
Different options
displayed
Figure B.6
appendix B | Thermal Cycler Programs 155
4.
Press F4 to select More.
Figure B.7
5.
Press F2 to select Insert.
Figure B.8
6.
Press F2 to select Cycle.
Do not choose Hold. Selecting Hold will not insert an additional hold step into the
program.
Figure B.9
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Now the generic program displays two cycle programs (3 Tmp 25 Cycles)
separated by one hold (1 Hld).
Figure B.10
Change the First Cycle Program to a Hold and Create the First 8 Holds
To change the first cycle program to a hold and set up the first 8 holds:
1.
Press the up arrow to highlight the 3 of the first 3 Temp 25 Cycles.
Figure B.11
2.
Using the keypad, change the 3 to a 1.
Figure B.12
3.
Press the down arrow.
appendix B | Thermal Cycler Programs 157
The wording in the display changes from 3 Temp 25 Cycles to 1 Hld.
Figure B.13
4.
Using the keypad, change the 1 to a 6.
Figure B.14
5.
Press the right arrow twice to move the highlight to the second hold program
(immediately adjacent to the first 6-stage hold just created).
Figure B.15
158
6.
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Using the keypad, change the 1 to a 2.
Figure B.16
Set the Times and Temperatures for the 6 Pre-PCR and 2 Hld
Programs
1.
Press the down arrow once, then the left arrow repeatedly to move from the 2 of 2
Hld back to the 94.0 of the 6 Pre-PCR program.
Figure B.17
2.
Using the keypad, change 94.0 to 58.0.
Figure B.18
appendix B | Thermal Cycler Programs 159
3.
Press the down arrow to highlight 0:30; then use the keypad to change 0:30 to
2:00.
Figure B.19
4.
Continue using the arrow keys and keypad to change each time and temperature
in the 6 Pre-PCR and 2 Hld programs to those shown below.
Note The last hold is not visible in the display as shown above.
You must use the right arrow key to move to the last hold
and set the temperature and time.
Figure B.20
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Set Up the Amplification Cycle
To set up the amplification cycle:
1.
Continue using the arrow keys and keypad to set the temperature and duration of
each PCR cycle as follows:
Table B.3
Temp of PCR Cycle
Temperature
Time
Temp 1 (Denature)
95°C
20 seconds
Temp 2 (Anneal)
64°C
45 seconds
Temp 3 (Extend)
72°C
10 seconds
Cycles
Meg 3-5-10k: 22 cycles
Meg 20k: 20 cycles
Note: Because the products being amplified are <120 bases in length, long extension times are not
required.
Program the Last Two Holds
To program the last two holds:
Continue using the arrow keys and keypad to advance to the ninth hold and set it
to 72°C for 10 seconds.
2. Advance to the last hold and set it to 4°C for Infinity.
1.
Store the Program
To store the program:
Press F2 to select Store.
2. If the User designation is:
1.
• Correct, proceed to the next step.
• Incorrect, press F2 and select the correct user.
3. Press F1 to select Accept.
4. Press F3 to select Method.
5. Press CE on the keypad to clear.
Use the arrows to move to the letter m, then press Enter on the keypad.
7. Continue entering the program name in the same manner (meg 3-5-10k or
meg 20k).
6.
• To select numbers (22 or 20), press the corresponding numbers on the keypad.
• To include a space, move to and enter the blank after the letter z.
Press F1 to select Accept.
9. Press F1 again to select Accept.
8.
appendix B | Thermal Cycler Programs 161
SET THE RAMP SPEED AND VOLUME
The ramp speed and volume must be set the first time you use the program. See
Setting the Ramp Speed and Volume for Each Program on page 148.
The ramp speed and volume for all of these programs are:
• Ramp speed
- GeneAmp PCR System 9700 with a gold or silver block = Max
- GeneAmp PCR System 9700 with an aluminum block = Std
• Volume: 67
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Meg Hypcr Thermal Cycler Programs
ABOUT THE MEG HYPCR PROGRAMS
Each of the Meg Hypcr programs consist of three holds and 1 cycle. The only
difference between these programs is the number of cycles. The version you will use is
determined by the assay panel size.
• For 3K and 5K assay panels, use Meg Hypcr 3-5k (9 cycles)
• For 10K and 20K assay panels, use Meg Hypcr 10-20k (10 cycles)
Ramp speeds:
• GeneAmp PCR System 9700 Thermal Cycler with a gold or silver block: Max
• GeneAmp PCR System 9700 Thermal Cycler with an aluminum block: Std
• GeneAmp PCR System 9700 Thermal Cycler with a dual block: 96D
• DNA Engine Peltier and Dyad: Gradient
Volume: 35 μL
The ramp speed and volume must be set the first time you use the program. See
Setting the Ramp Speed and Volume for Each Program on page 148.
Table B.4
Stages of the Meg Hypcr Thermal Cycler Programs
Stage
Temperature
Time
Denature
95°C
10 minutes
Denature
95°C
20 seconds
Anneal
68°C
45 seconds
Extend
68°C
10 seconds
Finish
68°C
10 seconds
Standby
4°C
Infinity
Cycles
3K/5K: 9 cycles
10K/20K: 10 cycles
appendix B | Thermal Cycler Programs 163
Figure B.21
Meg Hypcr Thermal Cycler Programs
Figure B.22P
Thermal Cycler Display for the Meg Hypcr 3-5k Program
Figure B.23
Thermal Cycler Display for the Meg Hypcr 10-20k Program
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Meg Hydigest-A Thermal Cycler Program
ABOUT THE MEG HYDIGEST-A PROGRAM
The Meg Hydigest-a program consists of three holds and no cycles.
Ramp speeds:
• GeneAmp PCR System 9700 Thermal Cycler with a gold or silver block: Max
• GeneAmp PCR System 9700 Thermal Cycler with an aluminum block: Std
• GeneAmp PCR System 9700 Thermal Cycler with a dual block: 96D
• DNA Engine Peltier and Dyad: Gradient
Volume: 75 μL
The ramp speed and volume must be set the first time you use the program. See
Setting the Ramp Speed and Volume for Each Program on page 148.
Table B.5
Stages of the Meg Hydigest-a Thermal Cycler Program
Stage
Temperature
Digestion
37°C
Time
30 minutes
60 minutes
Heat Inactivation
95°C
5 minutes
Standby
4°C
Infinity
If running a second QC gel, remove
4.0 μL of each sample when timer
reads 5:00 min remaining for the
second 37°C hold.
Figure B.24
Meg Hydigest-a Thermal Cycler Program
appendix B | Thermal Cycler Programs 165
Figure B.25
Thermal Cycler Display for Meg Hydigest-a Program
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Meg Denature Thermal Cycler Program
ABOUT THE MEG DENATURE PROGRAM
The Meg Denature program consists of two holds and no cycles.
Ramp speeds:
• GeneAmp PCR System 9700 Thermal Cycler with a gold or silver block: Max
• GeneAmp PCR System 9700 Thermal Cycler with an aluminum block: Std
• GeneAmp PCR System 9700 Thermal Cycler with a dual block: 96D
• DNA Engine Peltier and Dyad: Gradient
Volume: 90 μL
The ramp speed and volume must be set the first time you use the program. See
Setting the Ramp Speed and Volume for Each Program on page 148.
Table B.6
Stages of the Meg Denature Thermal Cycler Program
Stage
Temperature
Time
Denature
95°C
6 minutes
Standby
4°C
Infinity
Figure B.26M
Meg Denature Thermal Cycler Program
appendix B | Thermal Cycler Programs 167
Figure B.27
Thermal Cycler Display for the Meg Denature Program
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Appendix
C
Guidelines for Increasing Sample Throughput
170
Guidelines for Increasing Sample Throughput
This appendix provides guidelines for increasing sample throughput for the MIP
Assay Protocol from 48 to 96 samples per day based on a five day work week.
EQUIPMENT RECOMMENDATIONS
Increasing sample throughput based on the workflow recommended in Figure C.1 on
page 171 requires the following additional equipment. Refer to Chapter 2, Equipment,
Supplies, Consumables for part numbers.
• One additional thermal cycler in the Pre-Amp Lab (or an incubation block)
GeneAmp® PCR System 9700 Thermal Cycler
• One additional GeneChip® Hybridization Oven 640 with 8 carriers
• One to two additional GeneChip® Fluidics Station 450
RESOURCE REQUIREMENTS
Increasing sample throughput based on the workflow recommended in Figure C.1
requires one additional FTE (for a total of 3 FTEs). FTEs must be available to process
samples a minimum of 8 hours per day.
The process itself requires a minimum of 10 hours per day.
SUGGESTED WORKFLOW
The workflow shown in Figure C.1 on page 171 requires:
• That operators be proficient with running the MIP Assay Protocol.
• That every resource and all equipment is readily available and functional.
• A third thermal cycler or incubation block in the Pre-Amp Lab. This cycler is used
to hold the second round of annealed samples at 58°C until the thermal cyclers in
the Post-Amp Lab are available for use.
• Scanning 9 hours per day, assuming that new arrays are added to the Autoloader
every 30 minutes. In addition, the scanner must be left to run overnight.
appendix C | Guidelines for Increasing Sample Throughput 171
Figure C.1
Suggested Workflow for Processing 96 Samples Per Day
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Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
Appendix
D
Changing Plate Barcode Restrictions
174
How to Change Plate Barcode Restrictions
This appendix describes how to change the default barcode restrictions required by
Affymetrix GeneChip® Targeted Genotyping Analysis Software (GTGS). The default
barcode designations correspond to the names of the various plates used for sample
processing:
• Ann = Anneal Plate
• ASY = Assay Plate
• LBL = Label Plate
• Hyb = Hyb Plate
These designations help to ensure that the samples loaded onto each plate are properly
tracked throughout the MIP Assay Protocol.
The default barcode designations for plates help to ensure that the samples loaded
onto each plate are properly tracked throughout the MIP Assay Protocol. Therefore
we recommend that you do not change the default barcode designations.
To change the default barcode properties, modify the AssayTracking.properties file by
changing the designations highlighted below. The location of this file is:
C:\Program Files\Apache Group\Tomcat 4.1\webapps\geno\config\ Assay Tracking.properties
Modify only the text that is highlighted. Do not change the .*
# Defines plate tracking properties while performing assay
# Required barcode prefixes:
# Anneal plate:
ann-plate.pattern = ANN.*
ann-plate.errmsg = Barcode must start with ANN
# Assay plate:
asy-plate.pattern = ASY.*
asy-plate.errmsg = Barcode must start with ASY
# Label plate:
lbl-plate.pattern = LBL.*
lbl-plate.errmsg = Barcode must start with LBL
# Hybridization plate:
hyb-plate.pattern = HYB.*
hyb-plate.errmsg = Barcode must start with HYB
# Protocol:
protocol.pattern = PCL.*
protocol.errmsg = Barcode must start with PCL
appendix D | Changing Plate Barcode Restrictions 175
# Valid Thermal Cyclers (leave blank for no checking)
# use comma delimited integers for specific thermal cyclers (e.g. 1, 2, 3)
ann-plate.thermal-cyclers =
asy-plate.thermal-cyclers =
lbl-plate.thermal-cyclers =
hyb-plate.thermal-cyclers =
176
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
177
INDEX
A
about projects 141
activating users 133
adding array definitions 138
adding protocols 135
adding users 131
Affymetrix
contact information 144
Affymetrix GeneChip® Scanner 3000 Targeted
Genotyping System
system components 11
allele tube mixes
how to prepare 73
aluminum blocks
recommendation 12
amp mix
how to prepare 67
amp mix addition
about 60
how to perform 69
anneal cocktail
preparing 50
Anneal Plates
designing 40
viewing plate information 42
anneal stage
about 43
equipment and materials required 44
how to perform 44–52
kit components required 45
location and duration 44
Meg Anneal thermal cycler program 52
preparing anneal cocktail 50
sample concentration requirement 44
Applied Biosystems
contact information 144
array barcode
scanning 88
array definitions
adding 138
deleting 138
displaying array feature information 139
exporting 140
array features, exporting a list of 140
arrays
adding arrays while scanning 101
inspecting for and removing air bubbles 97
loading sample onto for hybridization 88
manually regridding 118
rescanning 103
rewashing 103
sample hybridization 86
scanning 100
assay panel files
about 25
deleting 106
importing 31
assay plates stage
about 53
equipment and materials required 53
planning and running 53–56
B
barcoded plates
part numbers and vendors 19
Bio-Rad Laboratories
contact information 145
Biorad precast gels and gel system 20
BioSmith Biotech
contact information 145
C
CEL files
troubleshooting missing files 115
centrifuge, mini
recommendation 12
centrifuge, plate
recommendation 12
clear film for arrays
part number and vendor 17
cleavage mix addition
about 59
how to perform 68
preparing the Cleavage Mix 67
Clontech
contact information 145
cluster genotype results
deleting 104
178
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
cocktail volumes, general information 23
contamination
preventing sample contamination 7
control DNA
about 8
benefits of using 8
in sample plate text files 30
recommendations 8, 22
Coriell Cell Repositories
contact information 145
Corning Conical Tubes
part number and vendor 18
equipment supplied by Affymetrix 11
required but not provided 12, 17
exo mix addition
about 58
how to perform 66
how to prepare exo mix 62
experiments
deleting 105
not hybridized 112
exporting a list of protocols 137
exporting a list of users 134
exporting project information 142
D
deactivating users 133
deleting
assay panel files 106
cluster genotype results 104
experiments 105
sample plates 106
deleting array definitions 138
deleting projects 142
deleting protocols 136
deleting users 132
design anneal plate stage 40
digest mix
how to add 79
displaying array definition details and features 139
dNTP addition
about 57
how to perform 65
dNTP plate
how to prepare 62
F
failed experiments
troubleshooting 124–128
first PCR
about 60
fluidics station
loading arrays onto 95
priming 93
shutting down 98
troubleshooting 108
freezer
recommendation 12
E
enzymes
storage conditions and recommendations 23
Eppendorf
contact information 145
Eppendorf Centrifuge 5804
part number 12
Eppendorf Color-Coded Safe-Lock Microcentrifuge
Tubes
part numbers and vendor 17
equipment and supplies required
vendor contact information 144
equipment, supplies & consumables required
G
gap fill mix addition
about 57
how to perform 62
how to prepare gap fill mix 61
gap fill, dNTP, ligate, invert and first PCR stage
about 57
equipment required 60
how to perform 57–69
kit components required 61
location and duration 60
other reagents required 61
preparing reagents 61
thawing reagents 61
gels
Biorad precast gels and gel system 20
Invitrogen precast gels and gel system 20
precast gel recommendations 20
QC recommendations 19
quality control recommendations 24
self-cast gel recommendations 20
Index 179
training requirements 19
GeneAmp PCR System 9700 Thermal Cyclers
part number 13
GeneChip SNP Kits 17, 22
contents and storage conditions 5
part numbers and descriptions 17
GeneChip Targeted Genotyping Analysis Software
description of 130
GeneChip Universal Tag arrays
part numbers 11
Gilson
contact information 145
GTGS
about projects 141
activating users 133
adding array definitions 138
adding protocols 135
adding users 131
array definition management 138
changing users 133
deactivating users 133
deleting array definitions 138
deleting projects 142
deleting protocols 136
deleting users 132
displaying array definition details and features
139
exporting a list of protocols 137
exporting a list of users 134
exporting array definitions 140
exporting array features 140
exporting project information 142
modifying array definition properties 139
modifying protocol properties 136
modifying users properties 134
project management 141
protocol management 135
H
hyb plates
how to make 78
Hybridization Cocktail
adding to samples 79
hybridization oven
speed required 89
temperature required 86
I
ice bucket & container recommendations 16
inversion of probe
about 59
Invitrogen Life Technologies
contact information 145
Invitrogen precast gels and gel system 20
L
label plates
how to make 72
ligation
about 57
M
Meg 20k thermal cycler program 152
Meg 3-5-10k thermal cycler program 152
Meg Anneal thermal cycler program 52, 150
Meg Denature thermal cycler program 166
running 87
Meg Digest thermal cycler program
running 80
Meg Hydigest-a thermal cycler program 164
Meg Hypcr programs 162
MicroAmp Clear Adhesive Films
part number and vendor 17
Microtube Tough Spots
part number and vendor 17
MIP Assay Protocol
list of stages 22
overview 3
mix volumes, general information 23
modifying protocol properties 136
modifying user properties 134
P
PCR 12-well Tube Strips
part number and vendor 17
pipet-aid, portable, recommendation 12
pipettes and pipette tips
list of validated pipettes and tips 14
pipettes, disposable
part numbers and vendor 17
pipetting tips 23
plates, 96-well barcoded
part numbers and vendors 19
preparation for sample hybridization stage
180
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
kit components required 85
location and duration 84
making hyb plates 78
transferring samples and adding hyb cocktail 79
projects
about 141
deleting 142
exporting project information 142
how to create 25
prerequisites 25
protocol, MIP Assay
list of stages 22
overview 3
protocols
adding 135
deleting 136
exporting a list of 137
how to manage 135
modifying protocol properties 136
Q
quality control gel, first
example 75
how to load 75
quality control gel, second
viewing 82
quality control gels
precast gel recommendations 20
recommendations 19
self-cast gel recommendations 20
training requirements 19
R
racks
96-place, recommendation 16
cube, recommendation 16
microtube, recommendation 16
preparation, recommendation 16
Rainin Instrument, LLC
contact information 145
reagent kits
see GeneChip SNP Kits
reagent reservoirs
part number and vendor 17
refrigerator
recommendation 12
regridding arrays manually 118
rescanning arrays 103
S
sample
transfer and split from anneal to assay plates 63
sample hybridization stage 87
about 84
equipment and materials required 84
how to perform 84–??
hybridization oven speed 89
hybridization oven temperature 86
loading sample onto arrays 88
preparing arrays 86
sample info files
about 25
adding to 27
creating 26
deleting unused sample info 28
updating 27
sample plate
adding to a project 38
sample plate text file
creating 29
sample plates
deleting 106
samples
concentration, extraction and purification
methods 2
criteria 2
guidelines for preventing contamination 7
normalize 22
requirements 2, 22
sources of genomic DNA 2
scan arrays stage
about 99
adding arrays while scanning 101
equipment and materials required 99
how to perform 99–102
location and duration 99
preparing the scanner 100
scanning arrays 100
shutting down the scanner 102
scanner
how to shutdown 102
preparing to scan 100
troubleshooting 111
second PCR stage
Index 181
about 70
adding PCR product and allele tube mixes to
label plates 73
equipment and materials required 71
how to perform 70–75
kit components required 71
location and duration 70
making label plates 72
other reagents required 71
preparing allele tube mixes 73
skipped experiments
identifying and resolving 122–123
SQL Server 2000 11
stain and wash stage
about 92
equipment and materials required 92
how to perform 92–98
kit components required 92
loading arrays on fluidics station 95
location and duration 92
priming the fluidics station 93
Storage Cocktail 94
Storage Cocktail
preparing 94
Stratagene
contact information 145
T
Taq DNA polymerase
Clontech 17
requirements 7
Stratagene 17
vendors and part numbers 7
target digest stage
about 77
adding digest mix 79
equipment and materials required 78
how to perform 77–80
kit components required 78
location and duration 77
running target digest thermal cycler program 80
second quality control gel 81
setting well status to fail 83
viewing second quality control gel 82
thermal cycler programs
Meg 20k program 64, 152
Meg 3-5-10k program 64, 152
Meg Anneal program 52, 150
Meg Denature program 87, 166
Meg Hydigest-a program 80, 164
Meg Hypcr programs 74, 162
Post-Amp Lab programs 148
Pre-Amp Lab programs 148
thermal cyclers
Applied Biosystems 13
DNA Engine by BioRad 13
list of validated thermal cyclers 13
timer, recommendation 16
troubleshooting
experiments not hybridized 112
fluidics station 108
missing .CEL files 115
scanner 111
U
USA Scientific, Inc.
contact information 145
users
activating 133
adding 131
changing without logging out 133
deactivating 133
deleting 132
exporting a list of 134
modifying user properties 134
user management 130
V
vendor contact information 144
vortexer
recommendation 12
VWR International
contact information 145
W
well status
setting to fail 83
workflow monitor button
using 113
182
Affymetrix GeneChip® Scanner 3000 Targeted Genotyping System User Guide
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