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SNP Genotyping

U S E R G U I D E

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Limited License and Disclaimer for Fluidigm Systems with Fluidigm IFCs

Except as expressly set forth herein, no right to copy, modify, distribute, make derivative works of, publicly display, make, have made, offer to sell, sell, use, or import a Fluidigm system or any other product is conveyed or implied with the purchase of a Fluidigm system (including the BioMark System, EP1 System, FC1 Cycler, or any components thereof), and Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs (integrated fluidic circuits/microfluidic chips with or without a carrier), IFC controller, software, reagents, or any other items provided hereunder. This limited license permits only the use by the buyer of the particular product(s), in accordance with the written instructions provided therewith in the User Guide, that the buyer purchases from Fluidigm or its authorized representative(s). Except to the extent expressly approved in writing by Fluidigm or its authorized representative(s), the purchase of any Fluidigm product(s) does not by itself convey or imply the right to use such product(s) in combination with any other product(s). In particular, (i) no right to make, have made, or distribute other instruments, Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs, software, or reagents is conveyed or implied by the purchase of the Fluidigm system, (ii) no right to make, have made, import, distribute, or use the Fluidigm system is conveyed or implied by the purchase of instruments, software, reagents, Digital Array IFCs from Fluidigm or otherwise, and (iii) except as expressly provided in the User Guide, the buyer may not use and no right is conveyed to use the Fluidigm system in combination with instruments, software, reagents, or Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs unless all component parts have been purchased from Fluidigm or its authorized representative(s). For example, purchase of a Fluidigm system and/or the IFC controller conveys no right or license to patents covering the Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs or their manufacture, such as 6,408,878, 6,645,432, 6,719,868, 6,793,753, 6,929,030, 7,494,555, 7,476,363, 7,601,270, 7,604,965, 7,666,361, 7,691,333, 7,749,737, 7,815,868, 7,867,454, 7,867,763; and EP Patent No. 1065378. Fluidigm IFCs may not be used with any non-Fluidigm reader, and Fluidigm readers may not be used with any chip other than Fluidigm IFCs. Fluidigm IFCs are single use only and may not be reused unless otherwise specifically authorized in writing by Fluidigm. All Fluidigm products are licensed to the buyer for research use only. The products do not have FDA or other similar regulatory body approval. The buyer may not use the Fluidigm system, any component parts thereof, or any other Fluidigm products in any setting requiring FDA or similar regulatory approval or exploit the products in any manner not expressly authorized in writing by Fluidigm in advance. No other licenses are granted, expressed or implied. Please refer to the Fluidigm website at www.fluidigm.com for updated license terms.

Fluidigm, the Fluidigm logo, BioMark, EP1, FC1, MSL, NanoFlex, Fluidline, Access Array, Dynamic Array, and Digital Array are trademarks or registered trademarks of Fluidigm Corporation in the U.S. and/or other countries. © Fluidigm Corporation. All rights reserved.

Limited Use License to Perform Pre-Amplification with Fluidigm Chips The purchase of Dynamic Array IFCs from Fluidigm Corporation conveys to the purchaser the limited, non-transferable right to perform pre-amplification methods under license from Life Technologies Corporation for use with the purchased amount of this product and Fluidigm instruments. No right to resell this product and no other rights (such as real-time PCR methods, apparatus, reagents or software to perform digital PCR methods) are conveyed by Life Technologies Corporation expressly, by implication, or by estoppel. For information on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008. For information about the scope of the above-identified Fields, please contact [email protected].

Fluidigm Product Patent Notice

Fluidigm products including IFCs (integrated fluidic circuits/microfluidic chips with or without a carrier) such as Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs, the IFC controller, FC1 Cycler and the Fluidigm system (BioMark System, EP1 System, readers, thermal cycler, etc.) and methods for reading and controlling the Access Array IFCs, Dynamic Array IFCs and Digital Array IFCs and/or their use and manufacture may be covered by one or more of the following patents owned by Fluidigm Corporation and/or sold under license from California Institute of Technology and other entities: U.S.

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Patent Nos. 6,408,878, 6,645,432, 6,719,868, 6,793,753, 6,929,030, 7,195,670, 7,216,671, 7,307,802, 7,323,143, 7,476,363, 7,479,186, 7,494,555, 7,588,672, 7,601,270, 7,604,965, 7,666,361, 7,691,333, 7,704,735, 7,749,737, 7,766,055, 7,815,868, 7,837,946, 7,867,454, 7,867,763, 7,906,072, 8,048,378; EP Patent Nos. 1065378, 1194693, 1195523 and 1345551; and additional issued and pending patents in the U.S. and other countries. Some Fluidigm IFC Controllers and associated IFCs may be licensed under Caliper Life Sciences. V5.6

© Fluidigm Corporation. All rights reserved.

No right to modify, copy, use, or distribute Fluidigm software is provided except in conjunction with the instrument delivered hereunder and only by the end user receiving such instrument in cases. Where the software and the associated instrument are beta test systems, NO WARRANTIES ARE PROVIDED, EXPRESSED OR IMPLIED. ALL WARRANTIES, INCLUDING THE IMPLIED WARRANTIES OF FITNESS FOR PURPOSE, MERCHANTABILITY, AND NON-INFRINGMENT ARE EXPRESSLY DISCLAIMED.

By continuing the installation process, user agrees to these terms. Please refer to the full text of the software license agreement supplied with the installation media for this application.

Every effort has been made to avoid errors in the text, diagrams, illustrations, figures, and screen captures. However, Fluidigm assumes no responsibility for any errors that may appear in this publication.

It is Fluidigm’s policy to improve products as new techniques and components become available. Therefore, Fluidigm reserves the right to change specifications at any time.

Information in this manual is subject to change without notice. Fluidigm assumes no responsibility for any errors or omissions. In no event shall Fluidigm be liable for any damages in connection with or arising from the use of this manual.

For Research Use Only. Not for use in diagnostic procedures.Contacting Fluidigm

For Technical Support

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Europe: [email protected]

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Phone

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Outside the United States: 650.266.6100

On the Internet: www.fluidigm.com/support

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Contents

Fluidigm SNP Genotyping User Guide v

Preface About this User GuideHow to Use This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Document Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Related Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Chapter 1 System OverviewGenotyping Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

System Components for Genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15IFC Controllers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Readers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Oven . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1648.48 Dynamic Array™ IFC - Genotyping . . . . . . . . . . . . . . . . . . . . . . . 17Reusable FR48.48 Dynamic Array™ IFC - Genotyping . . . . . . . . . . . . . . . 1796.96 Dynamic Array™ IFC - Genotyping . . . . . . . . . . . . . . . . . . . . . . . 18192.24 Dynamic Array™ IFC - Genotyping . . . . . . . . . . . . . . . . . . . . . . 18

Workflow for Standard IFCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Workflow for Reusable IFCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

What You Need for Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Supported Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20TaqMan® Assays and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20SNPtype™ Assays and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Supported Detection Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Probe Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Additional Probe Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

PCR Master Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Sample Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23DNA Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23DNA Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Using the Fluidigm FC1™ Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Powering On the FC1 Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Login . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Prepare Chip for Thermal Cycling . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Running a Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Cleaning Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

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Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Chapter 2 Using the Genotyping Analysis SoftwareLaunching the Genotyping Analysis Software . . . . . . . . . . . . . . . . . . . . . . 32

Open a Chip Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Finding Corners Manually (if required) . . . . . . . . . . . . . . . . . . . . . . . . . . 33Forced Manual Corner Find . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Setting Up Sample Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Using the Dispense Map Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Using Sample Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Using the Replay Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Setting Up Assay Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Changing Allele Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Advanced User Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Create a New Chip Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44New From Current Chip Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44Importing Multiple Chip Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Chapter 3 Viewing Run Data in the Analysis SoftwareViewing Data in Summary Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Using Cartesian Display/Polar Display . . . . . . . . . . . . . . . . . . . . . . . . . 50Changing the Confidence Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . 51Data Normalization Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Analyze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Details Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Results Table View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

All SNP Scatter Plot View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Image View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Call Map View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Using the Call Map View Zoom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58Changing the Call Map View Color Scheme . . . . . . . . . . . . . . . . . . . . . 59

Viewing Individual SNP Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

SNP Call View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Select Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Auto Scale/Full Range Toggle in SNP Call View . . . . . . . . . . . . . . . . . . 62Using Zoom In . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Using the Right-Click Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Changing Calls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65Show/Hide Invalid Option in SNP Call View . . . . . . . . . . . . . . . . . . . . . 67Highlight Selections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Experiment Information Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

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Using the Tool Menu Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70Results Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Generating Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Chip Run Preparation Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Chip Analysis Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Chip Run Performance Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Exporting Chip Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Chapter 4 Using the Assay Reference LibraryIntroduction to Assay Reference Library . . . . . . . . . . . . . . . . . . . . . . . . . 80

Setting Up Assay Reference Library in Toolbar Options . . . . . . . . . . . . . . . 80Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Using Assay Reference Library Extensions . . . . . . . . . . . . . . . . . . . . . . . . 81Assay Reference Library Main Menu . . . . . . . . . . . . . . . . . . . . . . . . . 81Assay Reference Library Manager . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Import Assays Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82Add Chip Run to Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Using Assay Reference Library to Analyze . . . . . . . . . . . . . . . . . . . . . . . . 85Update Embedded Assay Reference Library . . . . . . . . . . . . . . . . . . . . 87

Viewing Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Appendix A TaqMan® Assays for SNP Genotyping on the Dynamic Array IFCsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Required Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Required Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92Software Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Protocol for SNP Genotyping on 48.48 and 96.96 Dynamic Array IFCs . . . . . . 93Priming the 48.48 or 96.96 Dynamic Array IFC . . . . . . . . . . . . . . . . . . 93Preparing the 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . . 95Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96Using the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Protocol for SNP Genotyping on FR48.48 Dynamic Array IFCs . . . . . . . . . . . 99Priming the FR48.48 Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . 99Preparing the 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . .100Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . .101Using the FC1 Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .102

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Using the EP1 Reader Data Collection Software . . . . . . . . . . . . . . . . . 102

Protocol for SNP Genotyping on 192.24 Dynamic Array IFCs . . . . . . . . . . . . 104Preparing the 192.24 Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . 104Preparing the 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . 105Important Notes About Loading the 192.24 Chip . . . . . . . . . . . . . . . . . 105Using the FC1 Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107Using the EP1 Reader Data Collection Software . . . . . . . . . . . . . . . . . 108

Appendix B Convert Regular Chip Run to a More Samples Chip RunAccessing More Samples Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Set Up More Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Sample Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Assay Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Import the Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Appendix C SNPtype™ Assays for SNP Genotyping on the Dynamic Array™ IFCsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

Required Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116Required Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117Software Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

SNPtype Assays for SNP Genotyping on the 48.48 or 96.96 Dynamic Array IFCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Preparing the 10X SNPtype Specific Target Amplification (STA) Primer Pool for 48 assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118Preparing the 10X SNPtype Specific Target Amplification (STA) Primer Pool for 96 assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118Performing STA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Preparing SNPtype Assay Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Preparing 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . 120Priming and Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . 121Using the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . . . 124

SNPtype Assays for SNP Genotyping on the FR48.48 Dynamic Array IFC . . . . 126Preparing the 10X SNPtype Specific Target Amplification (STA) Primer Pool .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126Performing STA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126Preparing SNPtype Assay Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127Preparing 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . 128Priming and Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . 129Using the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . . . 131

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FR48.48 Dynamic Array IFC Cleaning Workflow . . . . . . . . . . . . . . . . . . . . .132Clean System and Interface Plate . . . . . . . . . . . . . . . . . . . . . . . . . . .132Initial Wash of the FR48.48 Dynamic Array IFC . . . . . . . . . . . . . . . . . .135Second Wash of the FR48.48 Dynamic Array IFC . . . . . . . . . . . . . . . . .136Remove Leftover Liquid from the FR48.48 Dynamic Array IFC . . . . . . . .136Dry the FR48.48 Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . .136Allow FR48.48 Dynamic Array IFC to Cool to Room Temperature . . . . . .137Reuse Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .138

SNPtype Assays for SNP Genotyping on the 192.24 Dynamic Array IFC . . . . . .139Preparing the 10X SNPtype Specific Target Amplification (STA) Primer Pool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .139

Performing STA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .139Preparing SNPtype Assay Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . .140Preparing 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . .141Priming and Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . .142Using the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . . . .149

Appendix D Fast Genotyping Using TaqMan Assays and the FC1 Cycler on the BioMark HD SystemIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152

Required Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152Required Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152Software Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152

Preparing 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .153

Preparing Sample Pre-Mix and Samples . . . . . . . . . . . . . . . . . . . . . . . . . .153

Priming and Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . .154

Fast Thermal Cycling the Dynamic Array IFC on the FC1 Cycler . . . . . . . . . .154

Fast Thermal Cycling the Dynamic Array IFC on the BioMark HD . . . . . . . . .155

Using the Data Collection Software for the EP1 Reader for an End-Point Read 156

Using the Data Collection Software for the BioMark System for an End-Point Read . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .157

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x Fluidigm SNP Genotyping User Guide

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Fluidigm SNP Genotyping User Guide 11

0About this User Guide 0

How to Use This GuideThe following chapters provide information about the analysis software and protocols for SNP genotyping on the BioMark™, BioMark HD or EP1 systems.

Document ConventionsThis guide uses specific conventions for presenting information that may require your attention. Please refer to the note conventions below.

CAUTION! This convention highlights potential bodily injury or potential equipment damage upon mishandling of the BioMark System. Read and follow instructions and/or information in a caution note very carefully to avoid any potential hazards.

WARNING! This convention highlights situations that may require your attention. May also indicate correct usage of instrument or software.

IMPORTANT: This convention highlights situations or procedures that are important to the successful outcome of your experiments.

NOTE: This convention highlights useful information.

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12 Fluidigm SNP Genotyping User Guide

Related DocumentsThis document is intended to be used in conjunction with these related documents:

• Fluidigm® Data Collection Software User Guide (PN 68000127)

• Fluidigm IFC Controller Usage Quick Reference (PN 68000126)

• Fluidigm 48.48 Genotyping Workflow Quick Reference (PN 68000099)

• Fluidigm 48.48 Fast Genotyping Workflow Quick Reference (PN 100-3882)

• Fluidigm 48.48 SNPtype Genotyping Workflow Quick Reference (PN 100-3910)

• Fluidigm 96.96 Genotyping Workflow Quick Reference (PN 68000129)

• Fluidigm 96.96 Fast Genotyping Workflow Quick Reference (PN 100-3909)

• Fluidigm 96.96 SNPtype Genotyping Workflow Quick Reference (PN 100-3912)

• Fluidigm 192.24 Genotyping Workflow Quick Reference (PN 100-3184)

• Fluidigm 192.24 SNPtype Genotyping Workflow Quick Reference (PN 100-3913) (PN 100-3913)

• Fluidigm Control Line Fluid Loading Procedure Quick Reference (PN 68000132)

•

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1System Overview 1

In this chapter:

Genotyping Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

System Components for Genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Thermal Cyclers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16

96.96 Dynamic Array™ IFC - Genotyping . . . . . . . . . . . . . . . . . . . . . . . .18

Workflow for Standard IFCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

Workflow for Reusable IFCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

What You Need for Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

Supported Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

SNPtype™ Assays and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Probe Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

Additional Probe Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

PCR Master Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

Sample Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

DNA Quality. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

DNA Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

Using the Fluidigm FC1™ Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

Powering On the FC1 Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26

Login . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

Prepare Chip for Thermal Cycling . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

Running a Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27

Cleaning Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30© Fluidig

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Genotyping OverviewGenotyping involves the analysis of variations in genomes across individual organisms. These variations often take the form of single nucleotide changes, known as single nucleotide polymorphisms, or SNPs, that can determine the characteristics and ultimate health of the organism. In SNP genotyping studies, the DNA sequences of a group of individuals are analyzed, often using a form of polymerase chain reaction (PCR), to determine which individuals have particular SNP profiles. Statistical analysis is then performed to determine whether a SNP or group of SNPs can be associated with a particular characteristic.

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System Components for Genotyping



IFC Controllers

IFC Controller HX

IFC Controller MX

IFC Controller WX Used for loading and cleaning of

IFC Controller RX

Used with 48.48 Dynamic Array IFCs for genotyping

and 12.765 and 48.770 Digital Array IFCs for dPCR

Used with 96.96 Dynamic Array IFCsfor genotyping and gene expression

FR48.48 Dynamic Array IFCs for Used with the 192.24 Dynamic Array IFCfor genotyping

genotyping

and gene expression

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Thermal Cyclers

Readers

Oven

NOTE: If you are using a Fluidigm Stand-Alone Thermal Cycler (not an FC1 Cycler), please see Appendix D for usage instructions or contact Fluidigm Technical Support for additional assistance.

FC1™ Cycler

EP1™ Reader BioMark™ or BioMark HD System

Convection oven (for FR48.48 Dynamic Array™ IFC)

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48.48 Dynamic Array™ IFC - Genotyping

Reusable FR48.48 Dynamic Array™ IFC - Genotyping

Sample inlets

Integrated fluidic circuit

Interface accumulator

Check valve

Containment accumulator

Check valve

Assay inlets

A1

Assay inlets

Integrated fluidic circuit

Interface accumulator

Check valve

Containment accumulator

Check valve

Sample inlets

A1 Note: Assay andSample inlets areopposite of standardDynamic Array IFCs

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Integrated

Sample inlets

Assay

Containmentaccumulator

Interfaceaccumulator

fluidic circuit

inlets

A1

Assay inlets

Sample inlets

Accumulator 2

A1

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Workflow for Reusable IFCs


Workflow for Standard IFCsThe simplicity of running experiments is illustrated in the process below. For more detail, see Fluidigm 48.48 Genotyping Workflow Quick Reference (PN 68000099), Fluidigm 96.96 Genotyping Workflow Quick Reference (PN 68000129), or Fluidigm 192.24 Genotyping Workflow Quick Reference (PN 100-3184).

1 Prime the genotyping IFC (Integrated Fluidic Circuit) (192.24 Dynamic Array IFC does not require priming).

2 Add the samples and assays to the IFC.

3 Load and mix samples and assays in the IFC Controller.

4 Perform PCR on the BioMark System or FC1 Cycler.

5 Collect images.

Workflow for Reusable IFCsThe simplicity of running experiments is illustrated in the workflow below. For more

details, see the FR48.48 Genotyping Quick Reference Cards, PN 100-2227 and PN 100-2228.

1 Prime the IFC in the IFC Controller (only required for first use).

2 Add samples and assays to the IFC.

3 Load the IFC in the Load IFC Controller.

4 Perform PCR on the BioMark System or FC1 Cycler.

5 Collect images.

6 Perform cleaning of the IFC in the Clean IFC Controller and reuse.

What You Need for ExperimentsThis section describes the materials that you need to perform your experiments including the reagents we support and sample requirements. If you deviate from the recommended procedures and material quantities, call Technical Support for help administering your experiments.

In addition, you need the following:

• BioMark or BioMark HD System or EP1 Reader with FC1 Cycler

• IFC Controller MX, HX, WX or RX (depending on IFC type)

• 48.48 Dynamic Array IFC–Genotyping, FR48.48 Dynamic Array IFC–Genotyping, 96.96 Dynamic Array IFC–Genotyping or 192.24 Dynamic Array IFC–Genotyping

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• Reagents

Supported ReagentsFluidigm currently supports Applied Biosystems TaqMan® reagents and Fluidigm SNPtype™ reagents.

TaqMan® Assays and Reagents

Applied Biosystems provides TaqMan® reagents for targeted and genome-wide discovery of single nucleotide polymophisms (SNPs), copy number variants, and other genetic variations.

The necessary reagents for TaqMan genotyping are as follows:

TaqMan protocols are included in Appendix B of this manual.


Store at -20°C

• TaqMan® Pre-Designed SNP Genotyping Assays (Applied Biosystems, PN 4351374)• AmpliTaq Gold® DNA Polymerase (Applied Biosystems, PN 4311806)*• 2X Assay Loading Reagent (Fluidigm, PN 85000736)• ROX reference dye 50X (Invitrogen, PN 12223-012)

Store at 4°C

• TaqMan® Universal PCR Master Mix (Applied Biosystems, 4304437)**• Genomic DNA• GT Sample Loading Reagent (Fluidigm PN 85000741)***

Store at room temperature

• Deionized DNA-free, DNase-free, RNase-free water• TE: 10 mM Tris HCl, 0.1 mM EDTA, pH 8.0 (Teknova PN T0221) (also known as DNA Suspension

Buffer).

* For reusable IFCs, do not use AmpliTaq Gold**For reusable IFCs or fast thermal cycling applications, do not use TaqMan Universal PCR mix, use GTXpress

Master Mix instead.*** For reusable IFCs or fast thermal cycling applications, do not use GT Sample Loading Reagent, use Fast

Sample Loading Reagent (Fluidigm, PN 100-3065) instead.

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Supported Reagents


SNPtype™ Assays and Reagents

Fluidigm SNPtype Assays are an allele-specific PCR detection system. It offers a low-cost, high-quality solution for SNP genotyping and are designed to work seamlessly with the EP1™, BioMark, or the BioMark™ HD System. It also provides the advantages of minimal experiment setup time and optimized protocols for Dynamic Array integrated fluidic circuits (IFCs).

Assays are provided in three separate oligo plates per 96 assays. All oligos are provided in nuclease-free water.

• Allele-Specific Primers (ASP)—mixed in equal molar ratios and normalized to 100 µM in 100 µL for small; 200 µL for medium; 500 µL for large (each primer)

• Specific Target Amplification (STA) primer—individual primers normalized to 100 µM in 100 µL for small; 200 µL for medium; 500 µL for large (each primer)

• Locus-Specific Primer (LSP)—individual primers normalized to 100 µM in 100 µL for small; 200 µL for medium; 500 µL for large (each primer)

SNPtype Assays use the same probe set in every reaction to enable low startup and low running costs, which are critical for high sample throughput applications such as production-scale SNP genotyping and SNP confirmatory studies. SNPtype Assays have been developed to provide excellent results on Dynamic Array IFCs and are formatted to work directly with existing workflows.

In conjunction with the FC1 Cycler, SNPtype Assays permit fast thermal cycling, allowing faster time-to-results and thereby greatly increasing sample throughput.

Protocols for genotyping with SNPtype Assays can be found in appendix C of this user guide.

The necessary reagents for SNPtype genotyping are as follows:

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Supported Detection ReagentsWe support the following genotyping detection reagents with the System.

Probe Types

For TaqMan

• FAM-MGB

Store at -20°C

• Biotium Fast Probe Master Mix (Biotium, PN 31005)• Qiagen 2X Multiplex PCR Master Mix (Qiagen, PN 206143)• SNPtype Genotyping Reagent Kit 48.48 (Fluidigm, PN 100-4135)

This kit is sufficient for ten 48.48 Dynamic Array IFCs.– 1 Assay Loading Reagent, 2X (1.5 mL)– 1 SNPtype Sample Loading Reagent, 20X (250 µL)– 1 SNPtype Reagent, 60X (70 µL)

• SNPtype Genotyping Reagent Kit 96.96 (Fluidigm, PN 100-4134)This kit is sufficient for ten 96.96 Dynamic Array IFCs.– 2 Assay Loading Reagent, 2X (1.5 mL)– 2 SNPtype Sample Loading Reagent, 20X (250 µL)– 2 SNPtype Reagent, 60X (70 µL)

• SNPtype Genotyping Reagent Kit 192.24 (Fluidigm, PN 100-4136)This kit is sufficient for ten 192.24 Dynamic Array IFCs.– 1 Assay Loading Reagent, 2X (1.5 mL)– 2 SNPtype Sample Loading Reagent, 20X (250 µL)– 2 SNPtype Reagent, 60X (70 µL)– 2 Pressure Fluid syringes (3.5 mL each)

• SNPtype Assays– SNPtype Assay Allele-Specific Primers (ASP) Plate (100 µM ASP1/100 µM ASP2)– SNPtype Assay Locus-Specific Primer (LSP) plate (100 µM )– SNPtype Assay Specific-Target Amplification (STA) primer plate (100 µM )

Store at 4ºC

• Genomic DNA

Store at Room Temperature

• DNA Suspension Buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA) (TEKnova, PN T0221)• PCR-certified water• 20 Control line fluid syringes (300 µL each) (from Fluidigm SNPtype Genotyping Reagent Kit

48.48)• 20 Control line fluid syringes (150 µL each) (from Fluidigm SNPtype Genotyping Reagent Kit

96.96)• 10 Control line fluid syringes (160 µL each) (from Fluidigm SNPtype Genotyping Reagent Kit

192.24)

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PCR Master Mixes


• VIC-MGB

For SNPtype

• SNPtype-FAM

• SNPtype-HEX

Additional Probe Types

Fluidigm does not support other probe types at this time, however, additional probe types may be run with the System using the following guidelines:

PCR Master MixesStandard TaqMan genotyping protocols use TaqMan® Universal PCR Master Mix (2X) (Applied Biosystems PN 4304437). Fast TaqMan genotyping protocols use GTXpress Master Mix. SNPtype genotyping protocols use Biotium Fast Probe Master Mix (Biotium, PN 31005). If you choose to use master mixes other than those listed above, you may have to alter the protocol described in this manual. Contact Fluidigm Technical Support for additional information.1

Sample Requirements

DNA Quality

Your DNA should have a 260:280 ratio between 1.5 and 1.8. Prior to use on an IFC, check the integrity of your DNA on a system such as the Agilent® 2100 bioanalyzer.

Fluorophores With...

Excitation Wavelengths Emission Wavelengths

between 465 and 505 nm And between 500 and 550 nm

between 510 and 550 nm And between 540 and 600 nm

1. Fluidigm recommends that you use onlyTaqMan® probes and/or other licensed PCR assay reagents from authorized sources. If you have any questions regarding whether you have a license to use particular reagents in PCR systems, you should contact the appropriate licensor and obtain clarification and their permission if necessary. For example, certain probes and their use may be covered by one or more patents held by Applied Biosystems and/or Roche Molecular Systems, which may be contacted at Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008or the Licensing Department, Roche Molecular Systems, Inc., 4300 Hacienda Drive, Pleasanton, CA 94588.

IMPORTANT: You must use a passive reference (ROX).

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For human DNA, we recommend using DNA concentrations of 60 nanograms per microliter or higher. If you intend to use lesser concentrations, contact Fluidigm Technical Support for advice.

We recommend performing specific target amplification (STA) on DNA derived from species with genomes significantly larger than human size.

DNA Storage

Avoid multiple freeze-thaw cycles by storing DNA at 4°C. For longer storage, aliquots may be stored at -20°C.

NOTE: In order for the analysis software to perform the auto-calling function, samples require at least one NTC normalized point.

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Using the Fluidigm FC1™ Cycler




LCD Touch Panel

IFC Locator

BioMark IFC

Lid

Chuck (not visible)

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Powering On the FC1 Cycler

1 If you are using a powerstrip, turn it ON.

2 Press the switch at the back of the FC1 Cycler to the ON position.

3 At completion of power up and instrument initialization, the Fluidigm FC1 Cycler home screen appears.

NOTE: The FC1 Cycler uses a touch-screen interface, therefore all interactions with the application occur by touching the screen.

NOTE: Initialization may take up to 1 minute to complete.

On (–) /Off (o)

Networkingport

Power cordinput socket

switch

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Login

1 Press Login button to log in.

2 Default password for the following users are:

Admin: use 123456

User: no password

Prepare Chip for Thermal Cycling

1 Remove the protective film from the bottom of the chip.

Running a Protocol

1 Press the Start button.

NOTE: For information on setting passwords, see the Fluidigm FC1 Cycler User Guide, PN 100-1279.

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2 Open the lid.

3 Place the chip onto the thermal cycling block (chuck) on top of the instrument by aligning the notched corner of the IFC chip to the A1 mark.

4 Close the lid.

5 Press Continue to display available thermal protocols.

6 Choose a protocol to run from the protocol selection window.

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7 Press Run.

8 Once the protocol is finished, a confirmation screen appears. (During an active protocol, Abort will cancel the chip run.)

Cleaning Protocol

It is important to keep the thermal chuck surface clean. Any grease or debris will impact the thermal contact between the chuck and chip. Turn off system prior to cleaning the chuck.

1 Turn system Off.Let system cool down.

2 Use a lint-free cloth and 70% isopropyl alcohol to gently wipe the chuck.

WARNING! Never press down on the chip when it is on the FC1 Cycler.

NOTE: A status screen appears with a time estimate for completion.

CAUTION! Make sure chuck has had time to cool. It can get very hot and cause burn injury.

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Troubleshooting

Error Message Possible Solutions

The screen is blank Check power connection at the wall outlet, power strip and instrument inlet.

Error while loading the chip, no vacuum

Verify the blue protective film has been removed from the bottom of the chip.

Verify the chip is properly seated.

Make sure the chuck surface and bottom of the chip are clean. Follow cleaning protocol above.

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2Using the Genotyping Analysis Software 2

In this chapter:Launching the Genotyping Analysis Software . . . . . . . . . . . . . . . . . . . . . . .32

Open a Chip Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .32

Finding Corners Manually (if required) . . . . . . . . . . . . . . . . . . . . . . . . . . .33

Setting Up Sample Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34

Using the Dispense Map Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37

Using Sample Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40

Using the Replay Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41

Setting Up Assay Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41

Changing Allele Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43

Advanced User Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44

Create a New Chip Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44

New From Current Chip Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44

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Launching the Genotyping Analysis Software1 Double-click the Genotyping Analysis software icon on the desktop.

You can either Create a New Chip Run or Open a Chip Run.

Open a Chip Run1 Click Open a Chip Run.

2 Navigate to the Genotyping folder for the run that you want to analyze.

3 Double-click the chip run file you want (.bml extension).

Chip Explorer pane

Task pane

Chip Run Summary pane

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Open a Chip Run


Finding Corners Manually (if required)

If this occurs, you can manually set the corners and then analyze the chip.

1 Click OK.

2 Zoom in to see the corner cells.

3 Position the corners of the red box at the perimeter of the chip image.

4 Make sure each corner is placed on the outer edges of each corner cell (see below).

5 Click Done.

NOTE: If the chamber finding algorithm cannot locate the four corner cells of the chip during the first analysis, the following error message will appear.

NOTE: If you cannot see the four corner cells, adjust the Contrast slider. (If an insufficient amount of ROX dye was used in setting up the IFC corners, it will be difficult to see the corner cells.

Upper left corner Upper right corner

Lower left corner Lower right corner© Flui

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Forced Manual Corner Find

If the automated manual corner find results are not satisfactory, you can perform a forced manual corner find by pressing the CTRL key and simultaneously clicking on the Analyze button.

Setting Up Sample InformationSet up your sample to match the actual sample plate you used in your test.

1 Click Sample Setup in the Chip Explorer pane.

2 Click New. Alternately, click Import to open a saved sample list (.plt or .csv extension). If the sample plate has been set up previously, an alert opens:

3 Click Yes.

NOTE: If no ROX is present, the corner cells are very dark. You may have to count the number of rows and columns (48 down, 48 across for the 48.48 IFC for example) to make sure you are placing cross hairs correctly. See figure below.

Align red box tolast row, eventhough there isno ROX there

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Setting Up Sample Information


4 Select container type and format.

5 Click OK.

6 Click the Sample Mapping button (see figure below). The Sample Mapping button is not available for all container types; only for SBS Plate.

7 From the file, select either Left or Right dispense map.

8 Highlight the cells to name using one of the following:

• Click and drag.

• Press CTRL while clicking individual cells.

• Click the corner square to select all cells.

NOTE: If you are analyzing a:- 48.48 chip run, select M48-Sample-SBS96 (left or right).dsp- FR48.48 chip run, select FR48-Sample-SBS96 (left or right).dsp- 96.96 chip run, select M96-Sample-SBS96.dsp- 192.24 chip run, select 192-Sample-SBS384.dsp.

Container type dropdown menu options:- SBS Plate: import custom plate with annotations that you have already completed from a previously saved run. Or, - Sample Inlets: select sample inlets to add annotations for the current run.

Container format: SBS96 is the default setting. If you choose Sample Inlets for type, format is inactivated. Use SBS384 for 192.24 Dynamic Array IFCs.

Sample Mapping File dialog

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9 Click Editor.

Click corner to select all.

Click to open the Sample Editor.

Highlightedcells.

Click here to select all

Light blue cells indicate dispense map. In this example, the left dispense map has been chosen.

Status bar.

Click the Sample Mapping button to open the mapping file.

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10 Select Unknown, NTC or Control from the Sample Editor menu.

11 Type the sample name.

12 Click Update. The highlighted cells reflect the changes.

Using the Dispense Map Editor

Use the Dispense Map Editor to set dispensing map parameters and to record custom load maps for future use. After recording your loading sequence, you can save it and play it back anytime.

1 Click Tools > Dispense Map Editor.

2 Click File > New.

3 Complete the New Dispense Map dialog using the following as a guide.

4 Click OK to open the new dispense map in the Dispense Map Editor.

NOTE: To save new settings, first analyze your data, then click File > Save. If you exit the application without saving, a warning appears.

Description of the experiment (optional)

Unique experiment name or chip barcode

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5 Click the Begin Editing button in the Recording Control pane.

a Click the first cell from the Source Plate. Then, click the location in the Target Plate.

b Continue clicking appropriate cells (that is to say, from the Source Plate to the Target Plate) until your custom loading map has been recorded.

6 Click Stop Editing.

NOTE: When you click Begin Editing, the dispensing pane becomes inactive.

Source Plate. Graphical representation of the plate from which the samples and/or assays are pipetted.

Dispense Map. This table show you where the samples and assays are on the chip.

Target Plate. This is a graphical representation of the plate into which the samples and/or assays are pipetted.

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Refer to the following two graphics as an example of custom loading and how it looks as you proceed.

2

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Review the loading pattern you have recorded by clicking the green arrow button in the playback control pane.

Using Sample MappingAfter setting up the sample plate, view and/or record the loading pattern in the Sample Mapping Viewer.

1 Click Sample Mapping View in the chip explorer pane.

2 Click a cell in the Source Plate to see where it loads on the Target Plate (see below).

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Using the Replay Control


Using the Replay ControlUse the replay controls to show you where and in what sequence the Target Plate receives the samples from the Source Plate.

Setting Up Assay InformationSet up your assay information to match the actual assay wells you used in your test.

1 Click Assay Setup.

2 Click New. Alternately, click Import to open a saved sample list (.plt or .csv extension). If the sample plate has been set up previously, an alert opens:

3 Click Yes.

Plays the sequence from start to finish, one row at a time. Click it once to pause and then click again to continue.

Moves the loading to the end position.

Advances the loading one row at a time with each click toward the end.Clears the map.

Returns the loading to the start

Load start position.

Load finish position.

Moves the loading back toward the start—one row at a time—with each click.

When you click Play, the yellow highlight moves sequentially from row 1 to row 6.

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4 Select container type and container format.

5 Click OK.

6 Click the Detector (Assay) Mapping button.

7 From the Sample Mapping file, select either Left or Right dispense map.

8 Highlight the cells that you are going to name using one of the following:

• Click and drag.

• Press CTRL while clicking individual cells.

• Click the corner square to select all cells.

9 Click Editor to launch the Assay Editor.

10 In the Assay Editor, type:

• Assay name

• Allele Y name

• Allele X name

NOTE: If you are analyzing a 48.48 chip run, you may use the left or right dispense map. If you are analyzing a 96.96 chip run, use M96-Sample-SBS96.dsp. If you are analyzing a 192.24 chip run, you may use the left or right dispense map.

Container type menu options:- SBS Plate: import custom plate with annotations that you have already completed from a previously saved run.Or, - Assay Inlets: select assay inlets to add annotations for the current run.

Container format: no choices, only 96 default. If you choose Assay Inlets for type above, format is inactivated.

Detector (Assay) Mapping File dialogAssay Mapping button

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Changing Allele Settings


11 Click Update.

12 Click Analyze. The highlighted cells reflect the changes.

Changing Allele Settings1 Click Change button.

2 Using the following as a guide when changing allele settings.

Use the Scatter Plot Axis (SCP) menu to change from X-axis to Y-axis and vice versa. The changes are reflected in the scatter plot view.

The Color dialog

Change colors from the dropdown palette or click More colors...

You can change the allele symbol here. If you fail to type something in one of the boxes, you are alerted by the exclamation point in a red sphere as shown above.

X

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Advanced User OperationsBelow are additional ways to load chip runs into the analysis software. These are generally used by advanced users.

Create a New Chip Run

1 Click Create a New Chip Run.

2 Follow the steps in the wizard to create a new chip run.

3 Proceed to “Setting Up Sample Information” on page 34.

New From Current Chip Run

To create a new chip run template using data from an analyzed chip that includes sample and assay settings, perform the following:

1 Open an analyzed and annotated chip run (.bml file extension).

2 Click File > New From Current Chip Run.

3 Open an unanalyzed and annotated chip run to create a run based on the analyzed run.

4 Type a name for the new chip run or, type the barcode number and then check Use Barcode as Chip Run Name.

5 Click Browse to navigate to a desired save location.

6 Click OK and the new chip run opens.

7 You can perform additional edits to the setup within the Genotyping software or save the file to be opened in the Data Collection software to run the new chip.

Importing Multiple Chip Runs

Import multiple chip runs to increase data points visible in the scatter plot view. Also, by importing multiple chip runs, more information is available for comparison when assessing questionable calls. Select up to eight chip runs to include in a multiple chip run.

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Advanced User Operations


1 Click File > Open Multiple Chip Runs.

2 Click the folder containing the multiple chip runs.

3 Click the arrow to move all the chip runs in the folder to the other pane. Alternately, expand the multiple chip run folder and choose specific .bml run files, clicking the arrow for each file that you want to add to the other pane. Alternatively, you can select files from various folders.

4 Type a name.

5 Click Browse.

6 Navigate to a location to store the data.

7 Click OK.

NOTE: There should be uniformity across the runs. For example, all should be end point runs or all should use the same thermal cycler protocol, etc.

Click the arrow to add runs

Type a name for the multiple run

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3Viewing Run Data in the Analysis Software 3

In this chapter:Viewing Data in Summary Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48

Changing the Confidence Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . .51

Data Normalization Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51

Analyze . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53

Details Views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53

Results Table View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53

All SNP Scatter Plot View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55

Image View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56

Image View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56

Using the Call Map View Zoom . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58

Changing the Call Map View Color Scheme. . . . . . . . . . . . . . . . . . . . . . .59

Viewing Individual SNP Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60

SNP Call View. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60

Using Zoom In . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63

Using the Right-Click Menus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64

Auto Scale/Full Range Toggle in SNP Call View. . . . . . . . . . . . . . . . . . . .62

Changing Calls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65

Show/Hide Invalid Option in SNP Call View . . . . . . . . . . . . . . . . . . . . . .67

Experiment Information Pane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69

Using the Tool Menu Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70

Results Export . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70

Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72

Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72

Generating Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73

Chip Run Preparation Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73

Chip Analysis Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74

Exporting Chip Run Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77

Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77

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Viewing Data in Summary Views1 Double-click the Genotyping Analysis software icon on the desktop to

launch the software.

2 Open a .bml chip run file and analyze if necessary (for more information, see “Launching the Genotyping Analysis Software” on page 32).

3 Click Summary Views (see figure below).

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4 Click a SNP of interest to open its scatter plot (see graphic below).

5 ([Optional] Press and hold the CTRL key while clicking multiple SNPs (see graphic).

NOTE: If you chose a 40-cycle protocol in the Data Collection Software, 40 images appear in the Summary Views. If you chose the End Point protocol, only one image appears.

Highlight a row and a corresponding scatter plot displays below.

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Using Cartesian Display/Polar Display

You can view the Scatter Plot data as a Cartesian Display or a Polar Display by clicking the Cartesian/Polar toggle button for individual SNPs and Summary View.

Cartesian Display

This is a standard view format for plotted results.

Each selected SNP displays a scatter plot. Use the horizontal scroll to see more scatter plots.

Multiple selected SNPs are highlighted and their scatter plots displayed.

The Play button allows you to step through the allele graphs.

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Polar Display

This view can help you to visually identify outlying data points.

Changing the Confidence Threshold

You can change the number in the Confidence Threshold text box (0-100) to reflect your desired level of confidence in the display of data points for a particular SNP assay. The confidence value of a particular clustering call (e.g. XX, XY or YY). It is calculated by examining the distance of the point to the center of the cluster it belongs to, along with the distances to the centers of the clusters it does not belong to. If a point is close to the center of the cluster it belongs to compared with the other cluster centers, it will have a higher confidence level.

If a data point call is less than the threshold number you set, the data point call is changed to No Call. The default confidence threshold setting is 65.

Data Normalization Method

Under Task, you can set the Data Normalization Method to “NTC” or “None.”

NTC stands for “No Template Control” normalization. This option makes viewing assays on the plotted graphs easier, because it normalizes the position of the no template control cells. The no template control cells are aligned to the x = .1 and y = .1 location on the plotted graph. It also normalizes the intensities of the assays so that they are roughly plotted in a square.

None does not normalize the NTC cells nor does it normalize the assays.

See examples below of NTC normalized data and non-normalized data.

Outlying data point

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Data Normalization Method: NTC

Notice the black no template control cells are shifted to the x = .1 and y = .1 location on the plotted graph

The assays intensities are plotted roughly in a square

Data Normalization Method: None

Note the no template control cells are not

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Details Views


Analyze

Be sure to click the Analyze button every time you change a parameter (see graphic below). A prompt in red text displays when you need to click the Analyze button again. A popup also displays.

Details ViewsWhen you click Details Views, three options are available:

• Results Table

• All SNP Scatter Plat View

• Call Map View

Each view opens data for the entire chip.

Results Table View

Without expanding it, click Results Table View to see the default view which includes data from the entire chip, each row representing a chamber on the chip.

NOTE: Click a single row or multiple rows to highlight them. Then, click Show Selected Rows to bring them to the top of the table. The selected rows remain highlighted.

In this example, changing the confidence threshold to 75 generates the red text prompt.

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Click Details Views to open default view

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All SNP Scatter Plot View


All SNP Scatter Plot ViewThe All SNP Scatter Plot View option shows all SNP data from the chip in one scatter plot. Use the zoom to enlarge areas of interest.

1. Click Zoom In to display the zoom map (left).2. Position your cursor over the highlighted square, click and drag the square to the area of interest.3. Click and drag the cursor around data points (right) or, alternately, click a point to highlight it.4. Change the call of the highlighted data point

Right-click a point of interest for more options

6

4

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Image ViewThe Image View option shows the inlets on the chip.

Right-click an inlet to display details.

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Call Map View


Call Map ViewThe Call Map View shows the location and call of each SNP on the chip.

Switch from Inlet-based View to Chip-based View to Custom View.Inlet-based View shows a map of the chip based on the delivery of assays and samples from the loading inlets on the chip.Chip-based View shows the actual physical position of the reaction on the chip.Custom View allows the user to define the layout of the Call Map View. It begins with the same layout as the Inlet-based View, but the user can select which rows and columns should be visible and the order of them through the column and row order buttons next to the layout selector.

Click and drag the column border to expand it.

Columnand roworder buttons

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Using the Call Map View Zoom

1. Click the Map icon to display the map.

2. Repeatedly click the Zoom in icon to enlarge the individual cells as shown below.

Highlight window

Hover over cells for more information

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Call Map View


Changing the Call Map View Color Scheme

1. Go to Grid Preferences > Preferences.

2. Click the color you want to change.

3. Click a basic color and then click OK.

5. Alternately, click Define Custom Colors to expand color choices.

6. Create a color on the right, and click Add to Custom Colors to display the new color in the grid.

7. Click the custom color in the grid and click OK.

The new color is reflected on the button.

4. Click OK to apply the color to the Call Map View.

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Viewing Individual SNP DataIndividual SNP data are shown, simultaneously, in the scatter plot pane, the Experiment Information pane, and the chip graphic.

SNP Call ViewThe SNP Call View shows specific SNP data as a scatter plot.

NOTE: Click to expand the Experiment Information pane.

NOTE: Click to expand the active pane. Click it again to return to the default view.

Scatter plot pane

Experiment information Chip graphic© Fluidig

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SNP Call View


Select Control

If you annotate any samples as “Control” types, a Select Controls button will appear. If no controls were selected, the button will not appear.

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Auto Scale/Full Range Toggle in SNP Call View

Auto Scale determines the best zoom factor to present all data points in the scatter plot. The invalid data points are not used in determining the zoom range.

Full Range shows the data points without zoom.

Full Range view

Auto Scale view

Hold cursor over data point for detailed information

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SNP Call View


Using Zoom In

The Zoom In feature allows you to zoom in on an area of interest.

1 Click the Zoom In tab.

2 You can position the white box around an area of interest, and the new view will display under SNP Call View.

Zoom Indialog

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Using the Right-Click Menus

Right-click a point of interest to open an options menu. Right-click menus throughout the application vary. This is one example:

If you right-click each SNP individually, you see this menu:

Copy and then paste individual data point information into Word or Excel.Some menus have the“Copy Scatter Plot View” option which allows you to copy and paste entire scatter plot into Word or Excel.

6

Highlights entire vertical column

Highlights entire horizontal row

Goes to SNP scatter plot

4

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SNP Call View


Changing Calls

Highlight a data point to change the current call. Call choices are color-coded in the legend. You can use the Undo Call and Redo Call buttons to revert a call back to its previous state.

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1. Click Auto Scale to enlarge the scatter plot.

2. Click the data point of interest. Alternately, if the cluster is still too tight to clearly distinguish the data point, click Zoom In and position and resize the highlight window over the cluster containing the data point.

Default SNP Call View.

Drag the highlight window to an area of

3. Ensure you have located the correct data point by placing your cursor over it to display relevant information.

4. Click the data point to activate it—when active, the data point is larger.

Auto calls are circles and are made by the softwareUser calls are diamonds and are set manually

5. Select the appropriate call from the legend—in this example below, Invalid is selected and the data point now reflects that change.

Right-click a data point for menu options:—Go To Image—takes you to the Image View—Copy—copies the data point information; can be pasted into Word or Excel—Copy Scatter Plot View—copies a picture of the current scatter plot; can be pasted into Word or Excel

The circled data point can be identified as invalid by its color, orange, and that it has been manually changed by its diamond shape.

6. Click to update the

Clear User Calls restores individual points. Click anywhere inside the scatter plot to restore data point to its original size, color, and position.Clear All User Calls restores all data points

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SNP Call View


Show/Hide Invalid Option in SNP Call View

NOTE: Changing a data point to invalid takes it out of use in the software call calculation. Unlike a No Call (an ambiguous call), which is still used by the clustering algorithm.

1. Click Auto Scale to enlarge the scatter plot. Further enlarge the area using the zoom in function (see, “Using Zoom In” on page 63).

2. Highlight (click or circle) a data point.

3. Click Invalid.

4. Click Hide or Show Invalid.

Click Hide Invalid and the highlighted data point is removed from the scatter plot.

Click Clear User Calls to restore data point. Then, click anywhere inside the scatter plot to restore the data point to its original size and position.

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Highlight Selections

The Highlight Selections/No Highlight Selections toggle button allows you to highlight points of interest.

1 Hold down your right-mouse button and draw a circle around a point or points of interest.

The selected points are enlarged.

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SNP Call View


Experiment Information Pane

In the screenshot below, note that the highlighted data point is shown relative to its position on the scatter plot and its sample and assay sources on the chip.

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Expanding the Experiment Information Pane

Using the Tool Menu OptionsSet export, report and database parameters using Options.

Results Export

1 Click Tools > Options.

Push-pinned panes concealed along border

Click to expand the pane vertically. For additional space, use the push-pins to collapse surrounding panes. An expanded pane with unused panes ‘pinned’ to the left side is shown above.

Experiment Information pane expanded vertically

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Using the Tool Menu Options


2 Click Results Export.

3 Use the default export destination or browse to another location.

4 Click Apply.

5 [Optional] If you are not changing the Report (graphs) options, click OK.

Post Export Command

If you want to export your results to an integrated database system use the Post Export Command.

1 Go to Tools > Options.

2 Check the Use Post Export Command option.

3 Then, browse to the database system in the Command box.

4 Click Apply.

5 Click OK.

The data will be exported as a .csv file to the database system you selected.

Choose a destination from the Default Folder menu. The browse icon is activated when you choose User Specified Folder.

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Report


2 Click Report to open options.

3 Use the default export destination or browse to another location.

4 Select graph options. When you export data that includes graphs or scatter plots, they reflect the options you choose here.

5 Click Apply, and then click OK.

Library


2 Click Library on the Options dialog to see database options.

NOTE: See Chapter 4 in this manual for more information about Assay Reference Library.

Choose a destination from the Default Folder menu. The browse icon is activated when you choose User Specified Folder.

Check the Enable Assay Reference Library option to enable the library feature.

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Generating Reports


3 Enter (or browse to) the location where you would like to store your assay database.

4 Click Apply.

5 Click OK.

Generating ReportsThere are three different reports are available depending on the type of chip run being opened: Chip Run Preparation Report, Chip Run Analysis Report and Chip Run Performance Report.

Chip Run Preparation Report

This report is available if you open an unanalyzed chip run file or, if you create a new chip run file. The report shows dispense mapping information, and is saved as an .htm file.

1 Click Report > Chip Preparation Report.

2 Click Save to save the Chip Preparation Report as an .htm file. The report opens automatically after you save it.

NOTE: If the Enable Assay Reference Library is not checked and the currently open chip run has not been analyzed with an assay reference library before, the Assay Reference Library-related GUI features are hidden.

The report name defaults to the chip barcode number with the report

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3 An example of a Chip Preparation Report is shown below.

Chip Analysis Report

This report is available when an analyzed chip run is opened or, when an unanalyzed chip run is analyzed. The report shows analysis data, and is saved as an .htm file.

1 Click Report > Chip Run Analysis Report.

The default report name (File name) is the chip run barcode.

2 Click Save to save the Chip Run Analysis Report as an .htm file. The report opens automatically after you save it.

An example of a Chip Run Analysis Report is shown below. The report includes individual scatter plots for each of the 48 reactions.

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Generating Reports


Individual scatter plots for each of the 48 reactions.

Data for each of the 48 reactions.

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Chip Run Performance ReportThis report is designed to test the call performance of a chip with a specific setup.

NOTE: The report results will not be accurate if the chip setup is not correct. Please contact Fluidigm Technical Support with any questions.

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Exporting Chip Run Data


Exporting Chip Run Data

Exporting Data

Export chip run data in a comma separated value format (.csv) that can be viewed in programs such as Microsoft® Excel. You can export data from:

• Summary Table View

• Detailed Table View

• Call Map View

1 Click File > Export.

2 Type a name for the file.

3 Select the appropriate type of view.

4 Click Save.

5 To open the saved data, navigate to the .csv file.

6 Double-click the .csv file to open it in a program such as Microsoft® Excel.

Below is an example of exported data in a .csv file, opened in an Microsoft® Excel spreadsheet.

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Double-click between columns to expand them.

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4Using the Assay Reference Library 4

In this chapter:Introduction to Assay Reference Library . . . . . . . . . . . . . . . . . . . . . . . . . .80

Setting Up Assay Reference Library in Toolbar Options. . . . . . . . . . . . . . . . .80

Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .80

Using Assay Reference Library Extensions . . . . . . . . . . . . . . . . . . . . . . . . .81

Assay Reference Library Main Menu . . . . . . . . . . . . . . . . . . . . . . . . . . .81

Assay Reference Library Manager. . . . . . . . . . . . . . . . . . . . . . . . . . . . .81

Import Assays Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82

Add Chip Run to Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84

Update Embedded Assay Reference Library . . . . . . . . . . . . . . . . . . . . . .87

Using Assay Reference Library to Analyze . . . . . . . . . . . . . . . . . . . . . . . . .85

Viewing Run Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .88

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Using the Assay Reference Library


Introduction to Assay Reference LibraryAssay Reference Library is a feature that advanced users can use to analyze a single chip run against a known Assay Reference Library. The Assay Reference Library is especially helpful when performing clustering analysis. The larger the Assay Reference Library, the better tool it will be for clustering analysis. The performance of the Assay Reference Library depends on adding only well curated and analyzed chip runs (chip runs that have been annotated correctly and had outliers removed).

Setting Up Assay Reference Library in Toolbar Options

Library

To enable Assay Reference Library extensions you must first select them.

To enable the extensions:

1 Select Tools > Options.

2 Click Library on the Options dialog box to see database options.

3 Enter (or browse to) the location where you would like to store your assay database. You can name your assay reference library “Assay_database_1”, for example.

4 Click Apply.

5 Click OK.

IMPORTANT: If the Enable Assay Reference Library is not checked, the related GUI features are hidden.

Check the Enable Assay Reference Library option to activate the feature.

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Using Assay Reference Library Extensions


Using Assay Reference Library ExtensionsThe Genotyping Analysis software provides several assay database extensions that allow you to cluster and label homozygous and heterozygous clusters using previously archived data in an assay reference library. These extensions include:

• Assay Reference Library Main Menu

• Assay Reference Library Manager

Assay Reference Library Main Menu

The Assay Reference Library main menu (see figure below) allows you to launch the Assay Reference Library Manager, Add a Chip Run to Library or Update Embedded Assay Reference Library. The Assay Reference Library main menu shows up when the Enable Assay Reference Library check box is checked on the Tools > Options > Library screen.

Assay Reference Library Manager

The Assay Reference Library Manager can be used to view individual Assay Reference Libraries based on the system and specific chip type.

Only one instance of the Assay Reference Library Manager can be open at a time.

The Assay Reference Library Manager loads the currently designated Assay Reference Library file. A progress bar pops up to display progress and allows you to cancel the loading process, if you choose.

1 Select Assay Reference Library Manager from the Assay Reference Library Main Menu. The Assay Reference Library Manager application opens.

2 See callouts below for a description of the various components of Assay Reference Library Manager.© Flui

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Import Assays Wizard

To import assays to the assay reference library, use the Import Assays Wizard.

1 Open the Import Assays Wizard by selecting File > Import Assays from the Assay Reference Library Manager page.

2 Enter (or browse to) an assay database that you wish to open.

The System ID filter can be used to view data from a particular system. If you select “All”, it displays all data from all systems.

The Chip Type drop-down menu is used to view chip types, either 48.48 or 96.96.

The Options menu contains assay database settings and allele settings.

List of assays in

The Search Assay Name text box allows you to search for an assay by name.The Delete button is used to delete the currently selected assay.

The File menu has a sub-menu item Import Assays. If you click on this it launches the Import Assays Wizard. You can also export reference library data to a .csv file.

The lower part of the view area is a chip run table of respective chip runs contained in the

Cluster points can be selected by using the lasso

The Cluster Plot displays the data for the selected assay. The cluster centers belonging to the same

The Cluster spreads of each cluster from all chip runs in the selected assay are shown as lightly

The Display button allows you to view a Cartesian or Polar view.

The Edit menu allows you to remove chip runs.

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Using Assay Reference Library Extensions


3 Click Next. A second Import Assays Wizard page appears.

4 Select Assays from the reference library listed on the left side of the dialog box and click the forward arrow to move them to the Current Assays box on the right side.

5 Click Finish.

The selected assays are added to the current assay reference library and the wizard closes.

NOTE: If you do not select an assay reference library location, an error icon appears. You may mouse-over the icon to receive more specific information about the error. The Next and Back buttons are enabled when a valid assay database location is entered.

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Add Chip Run to Library

1 Click the Add Chip Run to Library sub-item on the Assay Reference Library main menu. The Add Checked Assays in Chip Run to Assay Database dialog box launches.

2 Select the assays you wish to add to reference library by checkmarking them individually on the left pane or by checking Check All at the bottom of the list.

NOTE: If the assay reference library file is read-only, an error message appears and the assay dialog box does not open.

The Toggle Display allows you to turn on and off the open ellipses.

Select specific Assays you want to add to library. Cluster centers of the selected assay are displayed as crosses.

This adds checked assays to library.

Or, click Check All to select all assays to add to library.

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Using Assay Reference Library to Analyze


If an assay in the current chip run has been added to the currently selected assay reference library before, the respective check box is replaced with a lock icon to protect against accidental overwriting. If you want to remove this protection, use the “Unlock to Overwrite” toggle to change the overwriting state.

Using Assay Reference Library to AnalyzeThe performance of the database depends on adding only well curated and analyzed chip runs (chip runs that have been named correctly and had outliers removed). With assay reference library, you can add a single chip run and analyze it against a known database to determine what components the new chip run contains. The assay reference library is especially helpful when performing clustering analysis. The larger the library of representative data, the better tool it will be for clustering analysis.

NOTE: You can click Cancel on the loading bar if you choose to cancel adding the assays to the assay reference library.

Unlock to Overwrite toggle

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The Assay Reference Library allows you to analyze a chip run against a designated assay reference library.

1 In the Task pane, check Analyze with Assay Database. This allows a new chip run to be analyzed against the currently selected assay database. The label below the check box “Database: Embedded in this chip run” indicates the currently selected assay database was used for analyzing.

Once the chip run is analyzed against an assay reference library, a snapshot of the assay reference library is stored with the chip run. The label below the check box turns from “Library: (path to library)” to “Library: Embedded in this chip run.” This means the next time the Analyze button is clicked, the embedded assay reference library snapshot—instead of the currently selected assay reference library—is used to perform clustering and labeling of the data. However, repeatedly clicking the Analyze button causes the chip run to be re-analyzed with the embedded snapshot of the assay reference library. If you want to analyze against the current assay reference library, click on the Update Embedded Assay Reference Library option under the Assay Reference Library main menu. This triggers a re-analyze operation and updates the stored embedded assay reference library snapshot in the chip run with the current assay reference library.

IMPORTANT: An individual assay reference library can only be built from chip runs of a specific chip type (48.48 or 96.96) run on a single system.

NOTE: You must also check Enable Assay Reference Library on the Options dialog box (Tools > Options > Library) for this option to appear on the Task pane.

Check Analyze with Assay Reference Library.Label indicates an embedded assay will be used.

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Using Assay Reference Library to Analyze


Update Embedded Assay Reference Library

The Update Embedded Assay Database feature allows you to update the embedded assay database or “snapshot” of an existing analyzed database. In other words, you can work with a segment (snapshot) of a database and then synchronize it to the greater database when you are finished.

1 Select Update Embedded Assay Database from the Assay Database menu.

A warning screen will appear.

2 Click Yes to update the embedded assay library.

Update Embedded Assay Reference Library

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Viewing Run DataYou can use the same components of the Genotyping Analysis software to view run data when the assay reference library extensions are used. See Chapter 3 in this manual for more detail.

Some choices on the Details Views and Summary Views pages have changed, due to the database extensions, such as the following.

NOTE: If you have used the Assay Reference Library to analyze a chip run against an assay reference library, there are two additional columns on the Summary View table: Clustering Method and Concordance. See figure below.

4

A new check box related to assay reference library extensions appears on Summary Views and Details Views pages.

Analyze with Assay Reference LibraryLibrary: Embedded in this chip run.

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Viewing Run Data


Assay Setup and Sample Setup views are exactly the same as when no assay reference library is used. Refer to sections “Setting Up Assay Information” on page 41 and “Setting Up Sample Information” on page 34 to set up assays and samples when assay reference library extensions are used.

NOTE: Samples require at least one NTC normalized point.

Two additional columns appear if Assay Reference Library has been used.

The Clustering Method column displays only a scatter plot icon if the SNP has not been analyzed with an assay reference library. This column displays both a scatter plot icon and an assay database icon if the SNP has been analyzed against an assay reference library.

The Concordance column displays the consistency between the results when a library is used and when it is not used. The number is high for well separated clusters and low for assays that do not have good cluster separation.

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Fluidigm Genotyping User Guide 91

ATaqMan® Assays for SNP Genotyping on the Dynamic Array IFCs A

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .92

Protocol for SNP Genotyping on 48.48 and 96.96 Dynamic Array IFCs . . . . . . .93

Priming the 48.48 or 96.96 Dynamic Array IFC . . . . . . . . . . . . . . . . . . . .93

Preparing the 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94

Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . . .95

Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .96

Using the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . . . . .97

Protocol for SNP Genotyping on FR48.48 Dynamic Array IFCs. . . . . . . . . . . . .99

Priming the FR48.48 Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . .99

Preparing the 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . . 100

Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Using the FC1 Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Using the EP1 Reader Data Collection Software . . . . . . . . . . . . . . . . . . 102

Protocol for SNP Genotyping on 192.24 Dynamic Array IFCs. . . . . . . . . . . . . 104

Preparing the 192.24 Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . 104

Preparing the 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . . 105

Important Notes About Loading the 192.24 Chip . . . . . . . . . . . . . . . . . . 105

Using the FC1 Cycler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Using the EP1 Reader Data Collection Software . . . . . . . . . . . . . . . . . . 108

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92 Fluidigm Genotyping User Guide

IntroductionThe following document describes the protocol for using Applied Biosystems TaqMan® assays on 48.48, 96.96, FR48.48 and 192.24 Dynamic Array Integrated Fluidic Circuits (IFCs) for SNP genotyping.

Required Reagents

Standard SNP Genotyping

• TaqMan Universal Master Mix (Applied Biosystems, PN 4304437)

• 20X GT Sample Loading Reagent (Fluidigm, PN 85000741) or

• AmpliTaq Gold® DNA Polymerase (Applied Biosystems, PN 4311806)

Fast Genotyping

• GTXpress® Master Mix (2x) (Applied Biosystems, PN 4401892)

• 20X Fast GT Sample Loading Reagent (Fluidigm, PN 100-3065)

Both Standard and Fast Genotyping

• SNP Genotyping Assay Mix (80X) Applied Biosystems)

• 2X Assay Loading Reagent (Fluidigm, PN 85000736)

• 50X ROX™ reference dye (Invitrogen, PN 12223-012)

• genomic DNA

• DNA-free water

Required Equipment

• Fluidigm FC1 Cycler or Fluidigm Stand-Alone Thermal Cycler

• EP1 Reader or BioMark or BioMark HD system

• IFC Controller MX, HX, WX or RX

• 96-well plates

• 48.48, 96.96, Fr48.48 or 192.24 Dynamic Array IFCs (Fluidigm)

Software Requirements

SNP Genotyping Analysis Software and Fluidigm Data Collection Software.

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Protocol for SNP Genotyping on 48.48 and 96.96 Dynamic Array IFCs

Priming the 48.48 or 96.96 Dynamic Array IFC

1 Inject control line fluid into each accumulator on the chip.

2 Remove and discard the blue protective film from the bottom of the chip.

3 For 48.48 IFCs: Place into the IFC Controller MX, then run the Prime (124x) script to prime the control line fluid into the chip.For 96.96 IFCs: Place into the IFC Controller HX, then run the Prime (138x) script to prime the control line fluid into the chip.

Figure 1. 48.48 Dynamic Array IFC

CAUTION! Use the IFCs within 24 hours of opening the package.

CAUTION! Due to different accumulator volumes:

– only use 48.48 syringes with 300 µL of control line fluid for 48.48 Dynamic Array IFCs, and

– only use 96.96 syringes with 150 µL of control line fluid for 96.69 Dynamic Array IFCs.

CAUTION! Control line fluid on the chip or in the inlets makes the chip unusable.

CAUTION! Load the chip within 60 minutes of priming.

Inject Control Line Fluid

A1

Sample Inlets

Assay Inlets

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Figure 2. 96.96 Dynamic Array IFC

Preparing the 10X Assays

In a DNA-free hood, prepare aliquots of 10X assays using volumes in the table below (scale up appropriately for multiple runs).

Component Volume per Inlet (µL)

Volume per Inlet with Overage (µL)

Volume per 50 µL Stock

SNP Genotyping Assay Mix (80X*) (Applied Biosystems)

0.5 0.625 6.25

2X Assay Loading Reagent (Fluidigm, PN 85000736)

2.0 2.5 25.0

ROX reference dye (50X) (Invitrogen, PN 12223-012)

0.2 0.25 2.5

DNA-free water 1.3 1.625 16.25

Total Volume 4.0 5.0 50.0

* If you are using 40X SNP assays, double the volume of SNP assay mix and reduce the DNA-free water. For other starting concentrations of SNP assay mix, call Fluidigm Technical Support.


Sample inlets

Assay inlets

A1

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Preparing Sample Pre-Mix and Sample Mixes

1 Combine the components in the table below to make the Sample Pre-Mix and the final Sample Mixture.

2 In a DNA-free hood, combine the four Sample Pre-Mix components in a 1.5 mL sterile tube—enough volume to fill an entire chip. Aliquot 3.48 µL of Sample Pre-Mix for each Sample.

3 Remove the aliquots from the DNA-free hood and add 2.52 µL of genomic DNA to each, making a total volume of 6 µL in each aliquot.

ComponentVolume per

Inlet (µL)

Volume per Inlet with Overage

(µL)

Sample Pre-Mix for 48.48 (µL)

(60 reactions for ease of pipetting)



TaqMan® Universal PCR Master Mix (2X) (Applied Biosys-tesm, PN 4304437)

2.5 3.0 180.0 360.0

20X GT Sample Loading Reagent (Fluidigm, PN 85000741)

0.25 0.3 18.0 36.0

AmpliTaq Gold® DNA Polymerase (Applied Biosystems, PN 4311806)

0.05 0.06 3.6 7.2

DNA-free water 0.1 0.12 7.2 14.4

genomic DNA (added individually to Sam-ple Pre-Mix)

2.1 2.52

Total 5.0 6.0

NOTE: In order for the analysis software to perform the auto-calling function, samples require at least one NTC nominalized point.

Sam

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Figure 3. 48.48 Dynamic Array IFC Pipetting Map


Loading the Dynamic Array IFC

CAUTION! Vortex thoroughly and centrifuge all assay and sample solutions before pipetting into the chip inlets. Failure to do so may result in a decrease in data quality.

Assay Inlets Sample Inlets

A1

A1

ASSAY INLETS SAMPLE INLETS

ACCUMULATORS

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1 When the Prime (124x or 138x) script has finished, press Eject to remove the primed IFC from the IFC Controller MX or HX.

2 Pipette 4 µL of each assay into each assay inlet on the IFC.

3 Pipette 5 µL of sample into each sample inlet on the IFC.

4 Place the IFC into the IFC Controller MX or HX.

5 For 48.48: Run the Load Mix (124x) script to load the samples and assays into the IFC.For 96.96: Run the Load Mix (138x) script to load the samples and assays into the IFC.

6 Press Eject to remove the loaded IFC from the IFC Controller MX or HX.

7 Remove any dust particles or debris from the chip surface.

You are now ready for your chip run.

Using the Data Collection Software

1 Double-click the Data Collection Software icon on the desktop to launch the software.

2 Click Start a New Run.

3 Check the status bar to verify that the lamp and the camera are ready. Make sure both are green before proceeding.

4 Place the IFC into the reader.

5 Click Load.

6 Verify IFC barcode and IFC type.

a Choose project settings (if applicable).

b Click Next.

7 Chip Run file:

a Select New or Predefined.

b Browse to a file location for data storage.

c Click Next.

IMPORTANT: For unused sample inlets, use 3.48 µL of sample mix and 2.52 µL of water per inlet. For unused assay inlets, use 2.5 µL assay loading reagent, 0.25 µL ROX reference dye and 2.25 µL water per inlet.

CAUTION! While pipetting, do not go past the first stop on the pipette. Doing so may introduce air bubbles into inlets.

-5.0

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8 Application, Reference, Probes:

a Select Application Type: Genotyping.

b Select Passive Reference: ROX.

c Select probe types.

d Click Next.

9 Click Browse to find the appropriate thermal protocol file.

– For 48.48 IFC: GT 48x48 Standard v1.pcl

– For 96.96 IFC: GT 96x96 Standard v1.pcl

10 Confirm Auto Exposure is selected.

11 Click Next.

12 Verify the chip run information.

13 Click Start Run.

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Protocol for SNP Genotyping on FR48.48 Dynamic Array IFCs

Priming the FR48.48 Dynamic Array IFC

1 Perform system clean once a day.

2 Clean interface plate with DNAZap (Applied Biosystems, PN AM9890) and a clean wipe every other chip run.

3 Inject fluid from the yellow-banded syringe into the Interface accumulator on the chip.

4 Pipette 300 µL of Pressure Fluid into the P1, P2 and P3 wells on the chip.

5 Inject fluid from the clear-banded syringe into the Containment accumulator on the chip. (Only for first run.)


7 Place the chip into the Load IFC Controller WX, then run the Prime (168x) script to prime the chip.

Figure 5. FR48.48 Dynamic Array IFC

CAUTION! Do not inject any control line fluid into the Waste inlet. It must remain empty.

IMPORTANT: The FR48.48 sample inlets location is reversed from the 48.48 and 96.96 Dynamic Array IFCs.

Inject clear-banded syringe

A1

Assay Inlets

Sample InletsC

ontainment

Interface

Inject yellow-banded syringe

Waste P1

P2 P3

* Please note the location of the sample inlets is reversed from 48.48/96.96 Genotyping IFCs.

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0.5 0.625 6.25


2.0 2.5 25.0

ROX™ reference dye (50X) (Invitrogen, PN 12223-012)

0.2 0.25 2.5




ComponentVolume per

Inlet (µL)


(µL)



GTX Master Mix (2X) (Applied Biosystems, PN 4401892)

2.5 3.0 180.0

20X Fast GT Sample Loading Reagent

(Fluidigm, PN 100-3065)

0.25 0.3 18.0


genomic DNA (added individually to Sample Pre-Mix)

2.0 2.4

Total 5.0 6.0 210.0

Sam

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Pre

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Loading the Dynamic Array IFC

1 When the Prime (168x) script has finished, remove the primed IFC from the Load IFC Controller WX.

2 Pipette 4 µL of each assay into each assay inlet on the IFC.

3 Pipette 5 µL of sample into each sample inlet on the IFC.

4 Place the IFC into the IFC Controller WX.

5 Run the Load Mix (168x) script to load the samples and assays into the IFC.

6 When the Load Mix (168x) script has finished, remove the loaded chip from the Load IFC Controller WX.


8 Cover the inlets with tape prior to thermal cycling. Remove tape after reading on the EP1 Reader.



IMPORTANT: Make sure you thoroughly mix all assay solutions and all samples before pipetting into the chip inlets.Check that P1, P2, and P3 wells contain adequate levels of Pressure Fluid.Check that the Interface Accumulator contains an adequate level of DI water.Check that the Waste well is empty.Make sure the interface plate on the Load IFC Controller WX is clean and dust free before loading the IFC. You can use Scotch® tape to remove dust and debris. For unused sample inlets, use 3.6 µL of sample mix and 2.4 µL of water per inlet. For unused assay inlets, use 2.5 µL assay loading reagent, 0.25 µL ROX reference dye and 2.25 µL water per inlet.


CAUTION! Start the chip run on the FC1 Cycler within four hours of loading the IFC.

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Figure 2: FR48.48 Dynamic Array IFC Pipetting Map

Using the FC1 Cycler


2 Open the lid.

3 Place the chip onto the thermal cycling block (chuck) on the top of the instrument by aligning the notched corner of the IFC chip to the A1 mark.

4 Close the lid.


6 Choose the GT 48x48 Fast v1.pcl protocol from the protocol selection window.

7 Press Run.


Using the EP1 Reader Data Collection Software

1 Double-click the Data Collection Software icon on the desktop.


3 Check the status bar to verify that the lamp and camera are ready. Make sure both are green before proceeding.

CAUTION! Never press down on the chip when it is on the FC1 Cycler.


Sample Assay

Waste P1

P2 P3

Co

nta

inm

ent

Inte

rface

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4 Place the loaded chip into the reader.


b Click Next.

5 Click Load.


a Select Application Type—Genotyping.

b Select Passive Reference (ROX reference dye).


d Click Next.


8 Click Start Run.

The IFC cleaning protocol can be found in “FR48.48 Dynamic Array IFC Cleaning Workflow” on page 133 or in the Fluidigm FR48.48 Dynamic Array IFC Cleaning Workflow (PN 100-2228).

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Protocol for SNP Genotyping on 192.24 Dynamic Array IFCs

Preparing the 192.24 Dynamic Array IFC





CAUTION! Use the IFCs within 24 hours of opening the package.

CAUTION! Due to different accumulator volumes: only use syringes with 150 µL of control line fluid.

CAUTION! Control line fluid on the chip or in the inlets makes the chip unusable.





0.375 0.5 6.25


1.5 2.0 25.0

ROX reference dye (50X) (Invitrogen, PN 12223-012)

0.15 0.2 2.5




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Important Notes About Loading the 192.24 Chip

ComponentVolume per

Inlet (µL)


(µL)



GTXpressMaster Mix (2X) (Applied Biosystesm, PN 4401892)

1.5 2.0 480.0


(Fluidigm, PN 100-3065)

0.15 0.2 48.0


genomic DNA(added individually to Sample Pre-Mix)

1.2 1.6

Total 3.0 4.0


CAUTION! Thoroughly vortex and centrifuge all assay solutions and all samples before pipetting into the chip inlets.

IMPORTANT: For unused sample inlets, use 2.4 µL of sample mix and 1.6 µL of water per inlet. For unused assay inlets, use 2 µL assay loading reagent, 0.2 µL ROX reference dye and 1.8 µL water per inlet.

IMPORTANT: Make sure the interface plate on the IFC Controller RX is clean and dust free before loading the IFC. You can use Scotch® tape to remove dust and debris.

Sam

ple

Pre

-Mix

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1 Pipette 3 µL of each assay and 3 µL of each sample into the respective inlets on the chip.

2 Pipette 150 µL of Pressure Fluid into the P1, P2 and P3 wells.

3 Pipette 20 µL of Pressure Fluid into the P4 and P5 wells.

4 Blot carrier surface with a dry, lint-free cloth.

5 Place chip into the Load IFC Controller RX.

6 Using the IFC Controller RX software, run the Load Mix (166x) script to load the sample and assays into the chip.

7 When the Load Mix (166x) script has finished, eject the loaded IFC from the Load IFC Controller RX.




CAUTION! Start the chip run on the FC1 Cycler within four hours of loading the IFC.

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Using the FC1 Cycler


2 Open the lid.

3 Place the chip onto the thermal cycling block (chuck) on the top of the instrument by aligning the notched corner of the IFC chip to the A1 mark.

4 Close the lid.


6 Choose the GT 192x24 Fast v1.pcl protocol from the protocol selection window.

7 Press Run.

CAUTION! Never press down on the chip when it is on the FC1 Cycler.

Acc1

P1

P3

P4

P2

V V

P5

1 1 2

3

3

4

4

5

5

6

6

7

7

9

8 9 10 11 12

19 20 21 22 23 24

31 32 33

A1

10

ASSAY INLETS

SAMPLE INLETS

12

13

15

16

18

19

21

22

24

13 14 15 16 17 18

25 26 27 28 29 30

37 38 39 40 41 42

49 50 51 52 53 54

61 62 63 64 65 66

73 74 75 76 77 78

85 86 87 88 89 90

97 98 99 100 101 102

109 110 111 112 113 114

34 35 36

43 44 45 46 47 48

55 56

121 122 123 124 125 126

133 134 135 136 137 138

145 146 147 148 149 150

157 158 159 160 161 162

169 170 171 172 173 174

181 182 183 184 185 186

57 58 59 60

67 68 69 70 71 72

79 80 81 82 83 84

91 92 93 94 95 96

103 104 105 106 107 108

115 116 117 118 119 120

127 128 129 130 131 132

139 140 141 142 143 144

151 152 154153 155 156

163 164 165 166 167 168

175 176 177 178 179 180

187 188 189 190 191 192

Acc2

2

8

11

14

17

20

23

Second dispense of eight assays

Assay Loading Key

First dispense of eight assays

Third dispense of eight assays No assays - leave empty

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Using the EP1 Reader Data Collection Software

1 Double-click the Data Collection Software icon on the desktop.


3 Check the status bar to verify that the lamp and camera are ready. Make sure both are green before proceeding.

4 Place the loaded chip into the reader.


b Click Next.

5 Click Load.



b Select Passive Reference (ROX reference dye).


d Click Next.


8 Click Start Run.


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BConvert Regular Chip Run to a More Samples Chip Run B

In this Appendix:Accessing More Samples Templates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Assay Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Sample Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

Assay Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Import the Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

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Accessing More Samples Templates

Set Up More SamplesThe More Samples feature requires special sample and detector setup. Fluidigm provides setup templates in the Microsoft® Excel® file type. You can use the following workflow to set up your samples and detectors and convert them to .csv files and then annotate sample plates and detector plates in the analysis software.

Sample Setup1 On your BioMark or EP1 system computer, go to C:\Program Files\Fluidigm\

BioMarkGenotypingAnalysis\ApplicationData\FileFormats.

2 Open the file labeled “SamplePlateDefinitionForMoreS”.

– You may have to enable Active X. To do so:

• Click on the Options tab.

• Select “Enable this content” from the Microsoft Office Security Options dialog box.

• Click OK.

3 Edit the Microsoft Excel template to match your experiment.

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Sample Setup


4 Click Create Plate CSV File button.

A new tab labeled “CSV file” will be added to the Excel file (see screenshot below).

5 Open the new tab and double check your annotations.

6 Click Save to a CSV file button to save the file and select a convenient location for future retrieval.

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Assay Setup1 Next, open the file labeled “AssayPlateDefinitionForMoreS”.

2 Edit the Microsoft Excel file to match your experiment.

3 Click Create Plate CSV File button.

A new tab labeled “CSV file” will be added to the Excel file.

4 Open the new tab and double check your annotations.

5 Click Save to a CSV file button to save the file and select a convenient location for future retrieval.

Import the PlatesImport the Sample plate files.

1 Open the SNP Genotyping Analysis software.

2 Open a chip run that you wish to annotate.

3 Select Sample Setup in the Chip Explorer.

4 Click Import in the Task pane.

5 Browse to the location where you saved your sample plate.

6 Click Open.

Import the Assay plate files.

1 Open the SNP Genotyping Analysis software.

2 Open a chip run that you wish to annotate.

3 Select Assay Setup in the Chip Explorer.

4 Click Import in the Task pane.

5 Browse to the location where you saved your assay plate.

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Import the Plates


6 Click Open.

7 Go to the Analysis view and click the Analyze button on the Task pane.

8 Go to File > Convert to More Samples Chip Run...

Below is an example of a more samples chip run.

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CSNPtype™ Assays for SNP Genotyping on the Dynamic Array™ IFCs C

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

SNPtype Assays for SNP Genotyping on the 48.48 or 96.96 Dynamic Array IFCs . . . . . 117

Preparing the 10X SNPtype Specific Target Amplification (STA) Primer Pool for 48 assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Performing STA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Preparing SNPtype Assay Mixes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Preparing 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

Preparing Sample Pre-Mix and Sample Mixes . . . . . . . . . . . . . . . . . . . . . . 120

Priming and Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . 121

Using the Data Collection Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

SNPtype Assays for SNP Genotyping on the FR48.48 Dynamic Array IFC . . . . . . . . . . 126


Performing STA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119






FR48.48 Dynamic Array IFC Cleaning Workflow. . . . . . . . . . . . . . . . . . . . . 133

SNPtype Assays for SNP Genotyping on the 192.24 Dynamic Array IFC . . . . . . . . . . . 140


Performing STA 119






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IntroductionThe following document describes the protocol for using SNPtype assays on 48.48, 96.96, FR48.48 or 192.24 Dynamic Array Integrated Fluidic Circuits (IFCs) for SNP genotyping. Please refer to Appendix B for standard protocols not covered in this appendix.

Assays are provided in three separate oligo plates per 96 assays. All oligos are provided in nuclease-free water.

• Allele-Specific Primers (ASP)—mixed in equal molar ratios and normalized to 100 µM in 100 µL for small; 200 µL for medium; 500 µL for large (each primer)

• Specific Target Amplification (STA) primer—individual primers normalized to 100 µM in 100 µL for small; 200 µL for medium; 500 µL for large (each primer)

• Locus-Specific Primer (LSP)—individual primers normalized to 100 µM in 100 µL for small; 200 µL for medium; 500 µL for large (each primer)

Required Reagents• Biotium Fast Probe Master Mix (Biotium, PN 31005)

• Qiagen 2X Multiplex PCR Master Mix (Qiagen, PN 206143)

• DNA Suspension Buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA) (TEKnova, PN T0221)

• SNPtype Genotyping Reagent Kit 48.48 (Fluidigm, PN 100-4135)This kit is sufficient for ten 48.48 Dynamic Array IFCs.

– 1 Assay Loading Reagent, 2X (1.5 mL) (Fluidigm, PN 85000736)

– 1 SNPtype Sample Loading Reagent, 20X (250 µL) (Fluidigm, PN 100-3425)

– 1 SNPtype Reagent, 60X (70 µL)(Fluidigm, PN 100-3402)

– 20 Syringes of control line fluid (300 µL each)




– 2 SNPtype Reagent, 60X (70 µL) (Fluidigm, PN 100-3402)





– 2 SNPtype Reagent, 60X (70 µL) (Fluidigm, PN 100-3402)


– 2 Pressure Fluid syringes (3.5 mL each)

• SNPtype Assays

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SNPtype Assays for SNP Genotyping on the 48.48 or 96.96 Dynamic Array IFCs


– SNPtype Assay Allele-Specific Primers (ASP) Plate (100 µM ASP1/100 µM ASP2)

– SNPtype Assay Locus-Specific Primer (LSP) plate (100 µM)

– SNPtype Assay Specific-Target Amplification (STA) primer plate (100 µM)

• 50X ROX (Invitrogen, PN 12223-012)

• genomic DNA

• PCR-certified water

Required Equipment

• Fluidigm FC1 Cycler or Fluidigm Stand-Alone Thermal Cycler

• EP1 Reader

• IFC Controller MX, IFC Controller RX or Load IFC Controller WX and Clean IFC Controller WX

• 96-well plates

• 48.48 Dynamic Array IFC (Fluidigm, PN BMK-M-48.48) or

– 96.96 Dynamic Array IFC (Fluidigm, PN BMK-M-96.96 GT) or

– FR48.48 Dynamic Array IFC (Fluidigm, PN BMK-M-FR48.48) or

– 192.24 Dynamic Array IFC (Fluidigm, PN BMK-M-192.24 GT)


SNP Genotyping Analysis Software v.3.1.1 or higher and Fluidigm Data Collection Software v.3.1.1 or higher is required for this protocol.


NOTE: If you are using a Fluidigm Stand-Alone Thermal Cycler (SATC) (not an FC1 Cycler), please contact Fluidigm Technical Support for a Personal Card, which includes SNPtype scripts specific to the SATC.

NOTE: Specific target amplification (STA) is highly recommended for all plant samples and any potentially low quality DNA. The STA protocol described on page 118 is not required for SNPtype assays if the samples are of high quality and appropriate concentration (60 ng/µL of human genome size equivalent). If you want to perform STA, we recommend the minimum sample concentration to be 10 ng/µL.If you do not want to perform STA, proceed to page 119

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Preparing the 10X SNPtype Specific Target Amplification (STA) Primer Pool for 48 assays

Prepare the primer pool as described in the following table.

Preparing the 10X SNPtype Specific Target Amplification (STA) Primer Pool for 96 assays


Component Volume (µL) Final Concentration

100 µM SNPtype Assay STA Primer (for each of 48 assays)

2 (x 48 = 96 total) 500.0 nM

100 µM SNPtype Assay LSP (for each of 48 assays)

2 (x 48 = 96 total) 500.0 nM

DNA Suspension Buffer 208.0

Total 400.0



2 (x 96 = 192 total) 500.0 nM


2 (x 96 = 192 total) 500.0 nM


Total 400.0

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Performing STA

1 In a DNA-free hood, prepare aliquots of STA Pre-Mix using volumes in the table below (scale up appropriately for multiple runs).

2 In a 96-well PCR plate, combine 3.75 µL STA Pre-Mix with 1.25 µL of genomic DNA and mix well.

3 Thermal cycle using the following protocol:

4 Dilute the STA products 1:100 in DNA Suspension Buffer.

5 Store diluted STA products at -20ºC until ready to proceed.

Preparing SNPtype Assay Mixes

1 Prepare each SNPtype Assay Mix as described in the table below.

ComponentVolume

(µL)

STA Pre-Mix for 48.48 with Overage (µL)(60 reactions for ease of

pipetting)

STA Pre-Mix for 96.96 with Overage (µL)(120 reactions for ease of

pipetting)

Qiagen 2X Multiplex PCR Master Mix (Qiagen, PN 206143)

2.5 150.0 300.0

10X SNPtype STA Primer Pool

0.5 30.0 60.0

PCR-certified water 0.75 45.0 90.0

genomic DNA 1.25

Total 5.0 225.0 450.0

Hold 14 Cycles

Temperature 95ºC 95ºC 60ºC

Time 15 minutes 15 seconds 4 minutes


SNPtype Assay ASP1/ASP2 (100 µM each)

3.0 7.5 µM

SNPtype Assay LSP (100 µM)

8.0 20.0 µM


Total 40.0

STA

Pre

-Mix

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Preparing 10X Assays

1 In a DNA-free hood, prepare aliquots of 10X assays using volumes in table below (scale up appropriately for multiple runs).

2 Combine 2X Assay Loading Reagent with PCR-certified water to create the Assay Pre-Mix.

3 Combine 4 µL of Assay Pre-Mix + 1 µL of each individual SNPtype Assay Mix (as prepared in table above) for a total of 5 µL 10X Assay Mix.


1 Combine the Biotium Fast Probe Master Mix, 20X SNPtype Sample Loading Reagent, SNPtype Reagent, ROX and PCR-certified water to make the Sample Pre-Mix as described in the table below.

2 Combine 3.5 µL of Sample Pre-Mix with 2.5 µL of each genomic DNA (gDNA) to make a total of 6 µL of Sample Mix solution.

ComponentVolume per

Inlet (µL)

Assay Pre-Mix for 48.48 with Overage (µL)


Assay Pre-Mix for 96.96 with Overage (µL)



2.5 150.0 300.0


SNPtype Assay Mix 1.0

Total 5.0

NOTE: Excess assays can be stored at -20ºC for up to three weeks.

NOTE: If using STA, add 2.5 µL of 1:100 diluted STA product instead of gDNA.If STA is not used, the recommended DNA concentration is the genome copy number equivalent to 60 ng/µL or higher of human DNA.

Ass

ay

Pre

-Mix

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3 Vortex the Sample Mix for a minimum of 20 seconds, and centrifuge for at least 30 seconds to spin down all components.

Priming and Loading the Dynamic Array IFC

1 Inject control line fluid into each accumulator on the IFC (see Figure 1).

2 Remove and discard the protective blue film from the bottom of the IFC.

ComponentVolume per

Inlet (µL)




(120 for ease of pipetting)

Biotium 2X Fast Probe Master Mix (Biotium, PN 31005)

3.0 180.0 360.0

SNPtype 20X Sample Loading Reagent (Fluidigm, PN 100-3425)

0.3 18.0 36.0

SNPtype Reagent (Fluidigm, PN 100-3402)

0.1 6.0 12.0

ROX(Invitrogen, PN 12223-012)

0.036 2.2 4.3


genomic DNA 2.5

Total 6.0 210.0 420.0


NOTE: See the Appendix B for complete running instructions.

Sa

mp

le P

re-M

ix

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3 Place the IFC into the IFC Controller MX.

4 For the 48.48 Dynamic Array IFC: Run the Prime (124x) script.

– For the 96.96 Dynamic Array IFC: Run the Prime (138x) script.

5 Press Eject to remove the primed IFC from the IFC Controller MX.

6 Pipette 4 µL of appropriate 10X Assay Mix into each assay inlet on the IFC (see Figures 1 and 2).

7 Pipette 5 µL of appropriate Sample Mix into each sample inlet on the IFC (see Figures 1 and 2).

8 Place the IFC into the IFC Controller MX.

9 For the 48.48 Dynamic Array IFC: Run the Load Mix (124x) script to load the samples and assays into the IFC.

– For the 96.96 Dynamic Array IFC: Run the Load Mix (138x) script to load the samples and assays into the IFC.

10 Press Eject to remove the loaded IFC from the IFC Controller MX.

11 Place the IFC onto the FC1 Cycler or Fluidigm Stand-Alone Thermal Cycler.

12 For the 48.48 Dynamic Array IFC: Run thermal cycling protocol SNPtype 48x48 v1:

Thermal Cycling Conditions Temperature Time

Hot Start 1 cycle of: 95ºC 5 min

Touchdown (from 64.0ºC-61.0ºC, dropping 1ºC per cycle)

1 cycle of:

1 cycle of:

1 cycle of:

1 cycle of:

95ºC64ºC72ºC95ºC63ºC72ºC95ºC62ºC72ºC95ºC61ºC72ºC

15 sec45 sec15 sec15 sec 45 sec15 sec15 sec45 sec15 sec15 sec 45 sec15 sec

Additional PCR cycles 34 cycles of: 95ºC60ºC72ºC

15 sec45 sec15 sec

Cool 1 cycle of: 25ºC 10 sec


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Figure 1. 48.48 Dynamic Array IFC assay and sample inlets

Figure 2. 48.48 Dynamic Array IFC pipetting schema


Sample Inlets

Assay Inlets

A1

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– For the 96.96 Dynamic Array IFC: Run thermal cycling protocol SNPtype 96x96 v1:



Thermal Mix 1 cycle of: 70ºC25ºC

30 min10 min



1 cycle of:

1 cycle of:

1 cycle of:

1 cycle of:




15 sec45 sec15 sec



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5 Click Load.



b Click Next.

7 Chip Run file:



c Click Next.





d Click Next.


10 Click Next.


12 Click Start Run.

NOTE: Choose SNPtype-FAM and SNPtype-HEX for SNPtype assays.

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SNPtype Assays for SNP Genotyping on the FR48.48 Dynamic Array IFC

Preparing the 10X SNPtype Specific Target Amplification (STA) Primer Pool


NOTE: Specific target amplification (STA) is highly recommended for all plant samples and any potentially low quality DNA. The STA protocol decribed on page 126 is not required for SNPtype assays if the samples are of high quality and appropriate concentration (60 ng/µL of human genome size equivalent). If you want to perform STA, we recommend the minimum sample concentration to be 10 ng/µL.If you do not want to perform STA, proceed to page 127.



2 (x 48 = 96 total) 500.0 nM


2 (x 48 = 96 total) 500.0 nM


Total 400.0

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Performing STA










Component Volume (µL)STA Pre-Mix for FR48.48

with Overage (µL)(60 reactions for ease of pipetting)


2.5 150.0

10X SNPtype STA Primer Pool 0.5 30.0

PCR-certified water 0.75 45.0

genomic DNA 1.25

Total 5.0 225.0

Hold 14 Cycles




SNPtype Assay ASP1/ASP2 (100 µM each) 3.0 7.5 µM

SNPtype Assay LSP (100 µM) 8.0 20.0 µM


Total 40.0

STA

Pre

-Mix

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3 Combine 4 µL of Assay Pre-Mix + 1 µL of each individual SNPtype Assay Mix (as prepared in table above) for a total of 5 µL 10X Assay Mix.



2 Combine 3.5 µL of Sample Pre-Mix with 2.5 µL of each genomic DNA (gDNA) to make a total of 6 µL of Sample Mix solution.

ComponentVolume per Inlet

(µL)

Assay Pre-Mix for FR48.48 with Overage (µL)



2.5 150.0



Total 5.0



As

say

P

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ix

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1 Inject fluid from the yellow-banded syringe into the Interface accumulator on the IFC.

2 Inject fluid from the clear-banded syringe into the Containment accumulator on the IFC (first chip run only).

3 Pipette 300 µL of Pressure Fluid into the P1, P2 and P3 wells on the IFC.

4 Remove and discard protective blue film from the bottom of the IFC.

5 Place the IFC into the Load IFC Controller WX.


(µL)

Sample Pre-Mix for FR48.48 (µL)



3.0 180.0


0.3 18.0


0.1 6.0

ROX (Invitrogen, PN 12223-012

0.036 2.2


genomic DNA 2.5

Total 6.0 210.0

NOTE: Samples require at least one NTC normalized point.

NOTE: See Appendix B for complete running instructions.

Sam

ple

Pre

-Mix

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6 Run the Prime (168x) script (first chip run only).

7 Press Eject to remove the primed IFC from the Load IFC Controller WX.



10 Place the IFC into the Load IFC Controller WX.


12 Press Eject to remove the loaded IFC from the Load IFC Controller WX.

13 Cover inlets with Scotch® tape.


CAUTION! For the first use, load the IFC in the Load IFC Controller WX within 60 minutes after priming. For subsequent uses of the IFC, do not prime the IFC (skip steps 5 to 7 for the second through fifth uses).

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15 Run thermal cycling protocol SNPtype 48x48 v1:

Figure 4. FR48.48 Dynamic Array IFC assay and sample inlets




1 cycle of:

1 cycle of:

1 cycle of:

1 cycle of:




15 sec45 sec15 sec



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Figure 5. FR48.48 Dynamic Array IFC pipetting schema






5 Click Load.



b Click Next.

7 Chip Run file:



c Click Next.

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FR48.48 Dynamic Array IFC Cleaning Workflow






d Click Next.


10 Click Next.


12 Click Start Run.


Clean System and Interface Plate

1 Perform system clean on each IFC Controller WX once a day.

a Log in as User or Administrator.

b Press the Tools button.

c Select Clean System.

d Place cleaning plate on the tray and press Start System Cleaning.

e When the system purge is complete, eject the tray. Rinse the tray and let tray dry for next use.


NOTE: Clean the FR48.48 Dynamic Array IFCs within 2.5 hours of use. If you are unable to clean them within 2.5 hours, leave Scotch® tape on the inlets and place them with a damp paper towel in an air-tight plastic bag and store them in the refrigerator until you are able to clean them.

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2 Lift up on the red tab of the interface plate and pull out.

3 Clean the interface plate with DNAZap™ (Applied Biosystems, PN AM9890) and a clean wipe every other chip run.

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4 Clean the interface plate with Scotch™ tape every other chip run or as needed to remove any dust particles.

5 Check gaskets on opposite side of the interface plate to make sure they are in place.

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Initial Wash of the FR48.48 Dynamic Array IFC

1 Remove remaining liquid from P1, P2, and P3 wells.

2 Pipette 200 µL of Pressure Fluid into P1, P2, and P3 wells.

3 Pipette 20 µL of Wash Solution #1 into the assay wells.

4 Pipette 20 µL of Wash Solution #1 into the sample wells.

5 Place the FR48.48 Dynamic Array IFC into the Clean IFC Controller WX and run the Wash1 (168x) script. The wash will take approximately 50 minutes.

6 After the Wash 1 (168x) script has finished running, remove liquid from the Waste reservoir and any leftover liquid from sample and assay wells with a pipette.

Figure 6. FR48.48 Dynamic Array IFC

CAUTION! Do not inject any liquid into the Waste inlet. It must remain empty.

NOTE: Make sure there are no bubbles in the wells.

CAUTION! Do not use a vacuum to remove liquids as it can damage the chip.

Inject clear-banded syringe

A1

Assay Inlets

Sample InletsC

ontainment

Interface

Inject yellow- banded syringe

Waste P1

P2 P3

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Second Wash of the FR48.48 Dynamic Array IFC

1 Check that wells P1, P2, and P3 have enough reagent in them (~200 µL).

2 Pipette 30 µL of Wash Solution #2 into the assay wells.

3 Pipette 30 µL of Wash Solution #2 into the sample wells.

4 Place the FR48.48 Dynamic Array IFC into the Clean IFC Controller WX and run the Wash2 (168x) script. The wash will take approximately 30 minutes.

5 After the Wash2 (168x) script has finished running, remove liquid from the Waste reservoir and any leftover liquid from sample and assay wells with a pipette.

Remove Leftover Liquid from the FR48.48 Dynamic Array IFC

1 Place the FR48.48 Dynamic Array IFC into the Clean IFC Controller WX and run the Purge (168x) script. The blowout will take approximately 30 minutes.

2 After the Purge (168x) script has finished running, remove liquid from the Waste reservoir and any leftover liquid from P1, P2 and P3 wells with a pipette.

Dry the FR48.48 Dynamic Array IFC

CAUTION! Do not inject any liquid into the Waste inlet. It must remain empty.

NOTE: Make sure the oven is clean before inserting the IFCs for baking.

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1 Insert spring clips into the check valves to keep them open for the baking process.

2 Cover the IFCs with lint-free cloth wipes during baking.

3 Place the chip with spring clips into the oven and bake at 80ºC for 16 or more hours. (Bake for no more than 24 hours.)

4 Check to ensure there is no remaining liquid.

Allow FR48.48 Dynamic Array IFC to Cool to Room Temperature

1 Remove spring clips from the check valves.

2 Allow IFCs to cool to room temperature before using again.

NOTE: Control line fluid will be left inside the containment accumulator. You can store a cleaned chip at room temperature between uses. You must run all five uses within two months.

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SNPtype Assays for SNP Genotyping on the 192.24 Dynamic Array IFC

Preparing the 10X SNPtype Specific Target Amplification (STA) Primer Pool


Performing STA



NOTE: Specific target amplification (STA) is highly recommended for all plant samples and any potentially low quality DNA. The STA protocol described on page 140 is not required for SNPtype assays if the samples are of high quality and of appropriate concentration (60 ng/µL of human genome size equivalent). If you want to perform STA, we recommend the minimum sample concentration to be 10 ng/µL.If you do not wish to perform STA, proceed to page 141.



2 (x 24 = 48 total) 500.0 nM


2 (x 24 = 48 total) 500.0 nM


Total 400.0

Component Volume (µL)STA Pre-Mix for 192.24

with Overage (µL)(220 for ease of pipetting)


2.5 550.0

10X SNPtype STA Primer Pool 0.5 110.0


genomic DNA 1.25

Total 5.0 825.0

STA

Pre

-Mix

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3 Combine 3.2 µL of Assay Pre-Mix + 0.8 µL of each individual SNPtype Assay Mix (as prepared in table above) for a total of 4 µL 10X Assay Mix.

Hold 14 Cycles




SNPtype Assay ASP1/ASP2 (100 µM each)

3.0 7.5 µM

SNPtype Assay LSP (100 µM) 8.0 20.0 µM


Total 40.0


(µL)

Assay Pre-Mix for 192.24 with Overage

(µL) (30 reactions for ease of

pipetting)


2.0 48.0



Total 4.0

Ass

ay

Pre

-Mix

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2 Combine 2.6 µL of Sample Pre-Mix with 1.9 µL of each genomic DNA (gDNA) to make a total of 4.5 µL of Sample Mix solution.





(µL)


(240 for ease of pipetting)


2.25 540.0


0.225 54.0


0.075 18.0

ROX(Invitrogen, PN 12223-012)

0.027 6.48


genomic DNA 1.9

Total 4.5 630.0


Sam

ple

Pre

-Mix

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1 Inject control line fluid into accumulator 2 (Acc2).



4 Remove and discard the protective blue film from the bottom of the IFC.

5 Pipette 150 µL of pressure fluid into P1, P2 and P3. Tilt IFC so that the pressure fluid covers the entire well.

6 Pipette 20 µL of pressure fluid into P4 and P5.

7 Place the IFC into the IFC Controller RX.


9 Press Eject to remove the loaded IFC from the IFC Controller RX.


11 Run thermal cycling protocol SNPtype 192x24 v1:

NOTE: See the Fluidigm 192.24 Genotyping Workflow Quick Reference (PN 100-3184) for complete running instructions.




1 cycle of:

1 cycle of:

1 cycle of:

1 cycle of:




15 sec45 sec15 sec



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Figure 8. 192.24 Dynamic Array IFC Assay Loading Key

Acc1

P1

P3

P4

P2

V V

P5

1 1 2

3

3

4

4

5

5

6

6

7

7

9

8 9 10 11 12

19 20 21 22 23 24

31 32 33

A1

10

ASSAY INLETS

SAMPLE INLETS

12

13

15

16

18

19

21

22

24

13 14 15 16 17 18

25 26 27 28 29 30

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109 110 111 112 113 114

34 35 36

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55 56

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181 182 183 184 185 186

57 58 59 60

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163 164 165 166 167 168

175 176 177 178 179 180

187 188 189 190 191 192

Acc2

2

8

11

14

17

20

23

Second dispense of eight assays

Assay Loading Key

First dispense of eight assays

Third dispense of eight assays No assays - leave empty

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Figure 9. First six sample dispense steps on the left side of the 192.24

Step 1 Step 2 Step 3

1 2 3

13 14 15

25 26 27

37 38 39

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1 2 3

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101 102

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125 126

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149 150

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4

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LEFT side

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Figure 10. Next six sample dispense steps on the right side of the 192.24

Step 9Step 8Step 7

8 9

19 20 21

31 32 33

43 44 45

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67 68 69

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91 92 93

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187 188 189

11 12

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59 60

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83 84

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107 108

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131 132

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155 156

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179 180

190 191

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34

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78 9

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187 188 189

11 12

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107 108

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155 156

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78 9

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187 188 189

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155 156

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190 191

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7

Step 12Step 11Step 10

8 9

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187 188 189

11 12

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83 84

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131 132

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155 156

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190 191

10

34

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82

106

130

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7

Right side

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Figure 11. Next six sample dispense steps on the left side of the 192.24


1 2 3

13 14 15

25 26 27

37 38 39

49 50 51

61 62 63

73 74 75

85 86 87

97 98 99

109 110 111

121 122 123

133 134 135

145 146 147

157 158 159

169 170 171

181 182 183

5 6

16 17 18

29 30

40 41 42

53 54

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77 78

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101 102

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125 126

136 137 138

149 150

160 161 162

173 174

184 185 186

4

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172

1 2 3

13 14 15

25 26 27

37 38 39

49 50 51

61 62 63

73 74 75

85 86 87

97 98 99

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121 122 123

133 134 135

145 146 147

157 158 159

169 170 171

181 182 183

5 6

16 17 18

29 30

40 41 42

53 54

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101 102

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125 126

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173 174

184 185 186

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1 2 3

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121 122 123

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157 158 159

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181 182 183

5 6

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53 54

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125 126

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149 150

160 161 162

173 174

184 185 186

4

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172


1 2 3

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73 74 75

85 86 87

97 98 99

109 110 111

121 122 123

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145 146 147

157 158 159

169 170 171

181 182 183

5 6

16 17 18

29 30

40 41 42

53 54

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77 78

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101 102

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125 126

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149 150

160 161 162

173 174

184 185 186

4

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1 2 3

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25 26 27

37 38 39

49 50 51

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73 74 75

85 86 87

97 98 99

109 110 111

121 122 123

133 134 135

145 146 147

157 158 159

169 170 171

181 182 183

5 6

16 17 18

29 30

40 41 42

53 54

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77 78

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101 102

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125 126

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149 150

160 161 162

173 174

184 185 186

4

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1 2 3

13 14 15

25 26 27

37 38 39

49 50 51

61 62 63

73 74 75

85 86 87

97 98 99

109 110 111

121 122 123

133 134 135

145 146 147

157 158 159

169 170 171

181 182 183

5 6

16 17 18

29 30

40 41 42

53 54

64 65 66

77 78

88 89 90

101 102

112 113 114

125 126

136 137 138

149 150

160 161 162

173 174

184 185 186

4

28

52

76

100

124

148

172

LEFT side

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Figure 12. Last six sample dispense steps on the right side of the 192.24



8 9

19 20 21

31 32 33

43 44 45

55 56 57

67 68 69

79 80 81

91 92 93

103 104 105

115 116 117

127 128 129

139 140 141

151 152 153

163 164 165

175 176 177

187 188 189

11 12

22 23 24

35 36

46 47 48

59 60

70 71 72

83 84

94 95 96

107 108

118 119 120

131 132

142 143 144

155 156

166 167 168

179 180

190 191

10

34

58

82

106

130

154

178

192

78 9

19 20 21

31 32 33

43 44 45

55 56 57

67 68 69

79 80 81

91 92 93

103 104 105

115 116 117

127 128 129

139 140 141

151 152 153

163 164 165

175 176 177

187 188 189

11 12

22 23 24

35 36

46 47 48

59 60

70 71 72

83 84

94 95 96

107 108

118 119 120

131 132

142 143 144

155 156

166 167 168

179 180

190 191

10

34

58

82

106

130

154

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78 9

19 20 21

31 32 33

43 44 45

55 56 57

67 68 69

79 80 81

91 92 93

103 104 105

115 116 117

127 128 129

139 140 141

151 152 153

163 164 165

175 176 177

187 188 189

11 12

22 23 24

35 36

46 47 48

59 60

70 71 72

83 84

94 95 96

107 108

118 119 120

131 132

142 143 144

155 156

166 167 168

179 180

190 191

10

34

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82

106

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7

8 9

19 20 21

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43 44 45

55 56 57

67 68 69

79 80 81

91 92 93

103 104 105

115 116 117

127 128 129

139 140 141

151 152 153

163 164 165

175 176 177

187 188 189

11 12

22 23 24

35 36

46 47 48

59 60

70 71 72

83 84

94 95 96

107 108

118 119 120

131 132

142 143 144

155 156

166 167 168

179 180

190 191

10

34

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82

106

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78 9

19 20 21

31 32 33

43 44 45

55 56 57

67 68 69

79 80 81

91 92 93

103 104 105

115 116 117

127 128 129

139 140 141

151 152 153

163 164 165

175 176 177

187 188 189

11 12

22 23 24

35 36

46 47 48

59 60

70 71 72

83 84

94 95 96

107 108

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131 132

142 143 144

155 156

166 167 168

179 180

190 191

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78 9

19 20 21

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43 44 45

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79 80 81

91 92 93

103 104 105

115 116 117

127 128 129

139 140 141

151 152 153

163 164 165

175 176 177

187 188 189

11 12

22 23 24

35 36

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59 60

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83 84

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107 108

118 119 120

131 132

142 143 144

155 156

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190 191

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5 Click Load.



b Click Next.

7 Chip Run file:



c Click Next.





d Click Next.


10 Click Next.


12 Click Start Run.


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DFast Genotyping Using TaqMan Assays and the FC1 Cycler on the BioMark HD System D

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

Preparing 10X Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Preparing Sample Pre-Mix and Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Priming and Loading the Dynamic Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . 154

Fast Thermal Cycling the Dynamic Array IFC on the FC1 Cycler . . . . . . . . . . . . 154

Fast Thermal Cycling the Dynamic Array IFC on the BioMark HD. . . . . . . . . . . . 155

Using the Data Collection Software for the EP1 Reader for an End-Point Read . . 156

Using the Data Collection Software for the BioMark System for an End-Point Read157

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IntroductionThis protocol is intended for use with the Fluidigm FC1™ Cycler or the BioMark™ HD System and either 48.48 or 96.96 Dynamic Array™ Integrated Fluidic Circuits (IFCs) for SNP Genotyping. Following this protocol will reduce the cycling time to approximately 30 minutes for the 48.48 Dynamic Array IFC and approximately 70 minutes for the 96.96 Dynamic Array IFC.

Achieving these fast cycling times requires the Fluidigm FC1 Cycler or BioMark HD System, a new thermal cycling program and updated chemistry. The FC1 Cycler allows the user to select the ramp rate. For this protocol, use the Fast ramp rate of 5.5°C/sec.

We recommend the use of the TaqMan® GTXpress Master Mix from Applied Biosystems. This master mix has been optimized for use with fast thermal cycling protocols and it is also suitable for use with standard cycling protocols. The GTXpress Master Mix does not contain dUTP or UNG. Therefore, the UNG step has been eliminated from the cycling program. This master mix also uses an alternative Hot Start method to reduce the time to reactivate the enzyme.

Required Reagents

• TaqMan Genotyping Assays (Applied Biosystems)

• TaqMan GTXpress Master Mix (Applied Biosystems, PN 4401892)

• DNA Suspension Buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA) (TEKnova, PN T0221)

• 2X Assay Loading Reagent (Fluidigm, PN 85000736)

• 20X Fast GT Sample Loading Reagent (Fluidigm, PN 100-3065)

• ROX (50X) (Invitrogen, PN 12223-012)

• DNA-free water

Required Equipment

• Fluidigm FC1 Cycler or BioMark HD System

• IFC Controller MX (for the 48.48 Dynamic Array IFC) or IFC Controller HX (for the 96.96 Dynamic Array IFC)

• EP1™ Reader or BioMark System for end-point reads (if the FC1 Cycler is used)


SNP Genotyping Analysis Software v.3.0.2 or higher and Fluidigm Data Collection Software v.3.0.2 or higher is required for this advanced development protocol.

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Preparing 10X Assays1 In a DNA-free hood, prepare aliquots of 10X assays using volumes in the table

below (scale up appropriately for multiple runs).

Preparing Sample Pre-Mix and Samples1 Prepare a Sample Pre-Mix solution containing the GTXpress Master Mix and

20X Fast GT Sample Loading Reagent sufficient for the number and type of chips to be run.

The following table provides the component amounts for one 48.48 or one 96.96 chip.

These volumes include some overage to account for pipetting error.

2 In a DNA-free hood, combine the two Sample Pre-Mix components in a 1.5 mL sterile tube--enough volume to fill an entire chip. Aliquot 3.5 µL of the Sample Pre-Mix for each sample.

3 Remove the aliquots from the DNA-free hood and add 2.5 µL of gDNA to each, to make a total volume of 6 µL in each aliquot.


(µL)



SNP Genotyping Assay Mix (80X*) (Applied BioSystems)

0.5 0.625 6.25


2 2.5 25

ROX (50X)

(Invitrogen, PN 12223-012)

0.2 0.25 2.5


Total 4 5 50


Volume per Inlet with

Overage (µL)

Sample Pre-Mix for 48.48

(µL) (60 for ease of

pipetting)

Sample Pre-Mix for 96.96

(µL) (120 for ease of

pipetting)

2X GTXpress Master Mix

2.5 3.0 180.0 360.0


0.25 0.3 18.0 36.0

dH2O 0.17 0.2 12.0 24.0

genomic DNA 2.08 2.5

Total 5 6

Sam

ple

Pre

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3 Place the chip into the IFC Controller MX (for the 48.48 Dynamic Array IFC) or the IFC Controller HX (for the 96.96 Dynamic Array IFC).

4 Run the Prime (124x) script for the 48.48 Dynamic Array IFC or Prime (138x) script for the 96.96 Dynamic Array IFC.

5 Remove the primed chip from the IFC Controller.

6 Pipette 4 µL of Assay Mix into each assay inlet on the chip.

7 Pipette 5 µL of Sample Mix into each sample inlet on the chip.

8 Place the chip into the IFC Controller MX (for the 48.48 Dynamic Array IFC) or the IFC Controller HX (for the 96.96 Dynamic Array IFC).

9 Run the Load Mix (124x) script (for the 48.48 Dynamic Array IFC) or the Load Mix (138x) script (for the 96.96 Dynamic Array IFC) to load the samples and assays into the chip.

10 When the Load Mix script has finished, remove the loaded chip from the IFC Controller.

11 Remove any dust particles or debris from the chip surface using scotch tape.


Fast Thermal Cycling the Dynamic Array IFC on the FC1 Cycler

1 Press the START button.

2 Open the lid.

3 Place the chip onto the thermal cycling block (chuck) on top of the instrument by aligning the notched corner of the chip to the A1 mark.

4 Close the lid.

5 Press CONTINUE to display available thermal protocols.

6 Choose the appropriate protocol:

CAUTION! Vortex thoroughly and centrifuge all assay and sample solutions before pipetting into the chip inlets. Failure to do so may result in a decrease in data quality.


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Fast Thermal Cycling the Dynamic Array IFC on the BioMark HD


a For the 48.48 Dynamic Array IFC: GT 48x48 Fast v3.pcl.

b For the 96.96 Dynamic Array IFC: GT 96x96 Fast v3.pcl.

7 Press Run.

The chip is now ready for imaging on the EP1 Reader or the BioMark HD System.

Fast Thermal Cycling the Dynamic Array IFC on the BioMark HD

1 Remove the blue protective film from the bottom of the chip, if this was not done previously and start the BioMark HD Data Collection Software if this was not done earlier. Refer to the Fluidigm® BioMark™ HD Data Collection Software v3.0 User Guide (PN 100-2451) for details on using the BioMark HD instrument.

2 Click Start New Chip Run.

3 Place the chip into the loading position.

4 Click Load.

5 Verify chip barcode and chip type:

a Choose project settings (if applicable)

b Click Next.

6 Chip run file:



c Click Next.


a Select Application Type—Genotyping


c Select probe: Manually.

d Probe 1: FAM-MGB.

e Probe 2: VIC-MGB.

f Click Next.

8 Click Browse to find the thermal cycling protocol file. These are found in the GT folder. Cycling conditions are shown in the thermal cycling table below.

a For the 48.48 Dynamic Array IFC: GT 48x48 Fast v3.pcl.

b For the 96.96 Dynamic Array IFC: GT 96x96 Fast v3.pcl.

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10 Click Next.


12 Click Start Run.

Using the Data Collection Software for the EP1 Reader for an End-Point Read

1 Double-click the EP1 Data Collection Software icon on the desktop to launch the software.


3 Confirm that the camera and lamp are at temperature.

4 Place the chip into the EP1 Reader.

5 Click Load.

6 Verify chip barcode and chip type.


b Click Next.

7 Chip Run file:



c Click Next.





d Probe 1: FAM-MGB.

e Probe 2: VIC-MGB.

f Click Next.


48x48 GT 96x96 GT # of Cycles

Step Time Temp Time Temp

Hot MixN/A N/A 30 min 70ºC

N/A N/A 10 min 25ºC

Hot Start 120s 95ºC 120s 95ºC

Denature 2s 95ºC 2s 95ºC

Cycling Anneal/Extend 20s 60ºC 20s 60ºc 45

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Using the Data Collection Software for the BioMark System for an End-Point Read


10 Click Next.


12 Click Start Run.

Using the Data Collection Software for the BioMark System for an End-Point Read

1 Double-click the BioMark Data Collection Software icon on the desktop to launch the software.


3 Confirm that the camera and lamp are at temperature.

4 Place the chip into the BioMark.

5 Click Load.

6 Verify chip barcode and chip type.


b Click Next.

7 Chip Run file:



c Click Next.


a Select Application Type--Genotyping.



d Probe 1: FAM-MGB

e Probe 2: VIC-MGB.

f Click Next.

9 Click Browse to find the thermal cycling protocol file:

a For the 48.48 Dynamic Array IFC: GT End Point v1.pcl.

b For the 96.96 Dynamic Array IFC: GT End Point v1.pcl.


11 Click Next.


13 Click Start Run.

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Index

Aalleles

changing settings 43assays

setting up information 41

BBioMark System

components of 14Genotyping Analysis Software 31

Cchip run

analysis report 74analysis report, example of 75exporting data 76importing multiple runs 44

contacting Technical Support iii

Ddetector, see assay 41dispense map editor, using 37

FFluidigm

contacting iiiFluidigm reaction mix, preparing 25

GGenotyping Analysis Software

all SNP scatter plot view, details view 55allele settings change 43assay information set up 41auto scale 62call map view zoom feature 58changing calls 65chip run data export 76chip run preparation report 73color scheme changing of, call map view 59confidence threshold change 51data normalization method 51detailed table view 53

dispense map editor usage 37experiment information pane 69full range 62image view, details view 56importing multiple chip runs 44launching 32replay control usage 41report generation 73, 88sample information set up 85SNP call view 60SNP data, individual view 60summary views 48toolbar options 70viewing run data 47, 79

Pprobes, additional types of 23probes, supported types of 22

Rreagents

supported detection 21replay control, using 41

Ssample requirements

DNA quality 23DNA Storage 24

samplessetting up information for 85

supported detection reagents 21

TTechnical Support

contacting iii

VViews 53

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Fluidigm Corporation

World Headquarters7000 Shoreline Court, Suite 100South San Francisco, CA 94080 USA

Fluidigm Europe, BVLuna ArenaHerikerbergweg 2381101 CM Amsterdam ZuidoostThe NetherlandsTel: +31 (0) 20 578 88 53Fax: +31 (0) 20 203 11 11

Fluidigm France SarlLes Conquérants, Bat Kilimandjaro1 avenue de l’Atlantique91940 Les UlisTel : +33 (0)1 60 92 42 40Fax :+33 (0) 1 60 92 11 31 Fluidigm Singapore Pte. Ltd.Block 1026, #07-3532Tai Seng Avenue Singapore 534413SingaporeTel: +65 6858 7316Fax: +31 (0) 20 203 1111

Fluidigm Japan KKLuminous 2nd Floor, 15-19Nihonbashi-KodenmachoChuo-ku, Tokyo 103-0001 JapanTel: +81 3 3662 2150Fax: +81 3 3662 2154

Fluidigm (Shanghai) Instrument Technology Co., Ltd.Room 2907, B Building, Far East International PlazaNo. 317 XianXia Road, 200051Shanghai, ChinaTel. +86 21 3255 8368Fax +86 21 3255 8369

Technical Support send email to: [email protected]

Phone in United States: 1.866.FLUIDLINE (1.866.358.4354)

Outside the United States: 650.266.6100

On the Internet: www.fluidigm.com/support

Visit our website at www.fluidigm.com

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