Thermo Fisher Scientific PharmacoScan Assay 96-Array Format Automated Workflow User guide

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

PharmacoScan™ Assay 96-Array Format

Automated Workﬂow

USER GUIDE

for use with:

PharmacoScan™ 96F Array Plate

PharmacoScan™ Reagent Kit 96 Reactions

Applied Biosystems™ NIMBUS™ Target Preparation Instrument

Catalog Numbers 903160 and 913025

Publication Number MAN0018101

Revision D.0

Thermo Fisher Scientiﬁc Baltics UAB |

V.A. Graiciuno 8, LT-02241 |

Vilnius, Lithuania

Products:

PharmacoScan™ Reagent Kit 96 Reactions

Aymetrix Pte Ltd |

7 Gul Circle #2M-01 |

Keppel Logistics Building |

Singapore 629563

Products:

PharmacoScan™ 96F Array Plate

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE

LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR

ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0018101

Revision Date Description

D.0 29 April 2021 •Added options for using alternative 96-well round deepwell plates and 96-well PCR plates.

•Added Axiom™ 96 Consumables Kit for Applied Biosystems™ NIMBUS™ 2.0 v2.

•Changed method name to Axiom™ 96 Sample Automated Target Preparation Solution.

•Added optional Thermo Scientiﬁc™ Digital Microplate Shaker, set at 900 rpm for 10 minutes.

C.0 11 December 2019 Adding information for Windows™ 10 users.

B.0 24 January 2019 Correct mPCR gel image in the appendix.

A.0 09 November 2018 New publication.

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these

products, you accept the terms and conditions of all applicable Limited Use Label Licenses.

TRADEMARKS: All trademarks are the property of Thermo Fisher Scientiﬁc and its subsidiaries unless otherwise speciﬁed. Alpillo is

a trademark and brand of Alpaqua Engineering, LLC. Axygen, is a trademark of Axygen, Inc. Bel-Art, Cryo-Safe, and Scienceware

are trademarks or registered trademark of Bel-Art Products Corp. Bio-Rad, Microseal, DNA Engine Tetrad, and Hard-Shell are

registered trademarks of Bio-Rad Laboratories, Inc. Boekel Scientiﬁc and Jitterbug are trademarks of Boekel Scientiﬁc. Eppendorf

and Mastercycler are registered trademarks of Eppendorf AG. Hamilton, Microlab, NIMBUS, and CO-RE are owned and/or registered by

Hamilton Company in the U.S. and/or other countries. Microsoft, Windows, and Excel are either registered trademarks or trademarks of

Microsoft Corporation in the United States and/or other countries. PerkinElmer is a registeredtrademark of PerkinElmer, Inc. Pipet-Aid is

a registered trademark of Drummond Scientiﬁc Company. Rainin, Pipet-Lite, and Green-Pak are trademarks of Rainin Instrument, LLC.

SpectraMax is a registered trademark of Molecular Devices Corporation. Vortex-Genie is a registered trademark of Scientiﬁc Industries,

Inc.

Contents

■CHAPTER 1 Overview ............................................................. 11

Introduction ................................................................... 11

About the PharmacoScan™ Assay 96-Array Format Automated Workﬂow ............. 12

Assay highlights ........................................................... 13

Multi-plate workﬂows ........................................................... 13

Overview of the PharmacoScan™ Assay 96-Array Format Automated Workﬂow ........ 14

■CHAPTER 2 Prepare genomic DNA .............................................. 15

Sources of genomic DNA ....................................................... 15

General requirements ........................................................... 16

Special requirements ....................................................... 16

Evaluate the quality of genomic DNA with 1% agarose E‑Gel™.................. 17

Genomic DNA extraction and puriﬁcation methods ................................. 18

Clean up genomic DNA ........................................................ 19

Genomic DNA preparation ...................................................... 19

Genomic DNA input requirements ........................................... 19

Time required ............................................................. 19

Equipment, consumables, and reagents required .............................. 20

Thaw samples and controls ................................................. 21

Quantify and dilute test sample gDNA ........................................ 21

Aliquot the diluted samples and the controls .................................. 22

Freeze or proceed ......................................................... 23

GeneTitan™ Array Plate Registration ﬁle ........................................... 23

Create and save a GeneTitan™ Array Plate Registration ﬁle ...................... 24

■CHAPTER 3 Set up the Applied Biosystems™ NIMBUS™ Target

Preparation Instrument ............................................................ 25

Required materials ............................................................. 25

Plate centrifuge ............................................................ 25

Labware and consumables for multiplex PCR ................................. 26

Labware used on the deck .................................................. 27

PharmacoScan™ Reagent Kit 96 Reactions ................................... 32

Consumables required for target preparation .................................. 33

PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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Pipette tips ............................................................... 33

Axiom™ GeneTitan™ Consumables Kit ........................................ 34

Guidelines for use .............................................................. 37

Guidelines for handling plates and tubes ...................................... 37

Guidelines for a run on the NIMBUS™ Instrument .............................. 38

Thermal cycler recommendations and protocols ............................... 38

PCR plate type by thermal cycler for the mPCR step ........................... 39

Deck setup for reagent tubes .................................................... 40

Cooling block template ..................................................... 40

Assemble the cooling block template with reagent tubes and tube collar .......... 40

■CHAPTER 4 Multiplex PCR and target preparation with the Applied

Biosystems™ NIMBUS™ Target Preparation Instrument ....................... 42

Stage 1A: Perform multiplex PCR (mPCR) ......................................... 43

Time required ............................................................. 43

Input required ............................................................. 43

Materials, labware, and reagents required ..................................... 44

Prepare for mPCR ......................................................... 45

Prepare the mPCR Master Mix .............................................. 46

Set up the mPCR Reaction Plate ............................................ 46

Run the PharmacoScan mPCR thermal cycler protocol ......................... 47

Store the mPCR Product Plate .............................................. 47

Workﬂow for Stage 1A: Multiplex PCR ........................................ 48

Stage 1B: Amplify the genomic DNA ............................................. 50

Time required ............................................................. 50

Input required ............................................................. 50

Materials, labware, and reagents required ..................................... 50

Perform the prerun checklist ................................................ 51

Prepare for DNA ampliﬁcation ............................................... 52

Run the DNA ampliﬁcation step .............................................. 53

Workﬂow for Stage 1B: DNA ampliﬁcation .................................... 58

Stage 2: Fragment and precipitate the DNA ....................................... 61

Time required ............................................................. 61

Input required ............................................................. 61

Materials, labware, and reagents required ..................................... 61

Perform the prerun checklist ................................................ 63

Prepare for fragmentation ................................................... 64

Run the fragmentation step, then precipitate samples .......................... 65

Workﬂow for mPCR spike-in to Ampliﬁcation Plate ............................. 70

Workﬂow for Stage 2: Fragmentation ......................................... 71

Contents

4PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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Stage 3: Centrifuge and dry pellets ............................................... 75

Time required ............................................................. 75

Input required ............................................................. 75

Equipment and consumables required ........................................ 75

Centrifuge and dry the pellets ............................................... 75

Stage 4A and 4B: Resuspension and hybridization preparation ...................... 77

Time required ............................................................. 77

Input required ............................................................. 77

Materials, labware, and reagents required ..................................... 77

Thaw reagents ............................................................ 79

Stage 4A: Prepare the resuspension buer ........................................ 80

Frozen pellets and Axiom™ Resusp Buer ..................................... 80

Perform the prerun checklist ................................................ 80

Prepare resuspension reagents .............................................. 81

Run the resuspension step .................................................. 82

Resuspend the samples by o-deck shaking .................................. 85

Workﬂow for Stage 4A: Resuspension ........................................ 86

Stage 4B: Prepare Hybridization Master Mix ....................................... 87

Perform the prerun checklist ................................................ 87

Prepare hybridization preparation reagents .................................... 88

Run the hybridization preparation step ....................................... 88

Workﬂow for Stage 4B: Hybridization preparation .............................. 92

Stage 4C: Perform sample QC ................................................... 94

Time required ............................................................. 94

Input required ............................................................. 94

Materials, labware, and reagents required ..................................... 94

Perform the prerun checklist ................................................ 95

Prepare the Hyb-Ready Plate ................................................ 96

Prepare gel diluent ......................................................... 97

Run the sample QC step .................................................... 97

Workﬂow for Stage 4C: Sample QC ......................................... 100

Stage 5: Prepare the hybridization tray ........................................... 102

About Stage 5: Prepare the hybridization tray ................................ 102

Time required ............................................................ 102

Input required ............................................................ 102

Materials, labware, and reagents required .................................... 103

Perform the prerun checklist ............................................... 103

Sample plate and array plate preparation .................................... 104

Prepare the GeneTitan™ MC Instrument ..................................... 105

Denature the samples ..................................................... 106

Run the prepare hybridization tray step ...................................... 107

Contents

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Load the hybridization tray and array plate into the GeneTitan™ MC Instrument ... 109

Workﬂow for Stage 5: Prepare the hybridization tray .......................... 109

Stage 6: Prepare GeneTitan™ reagent trays ....................................... 110

Time required ............................................................ 110

Materials, labware, and reagents required .................................... 111

Notes on handling reagents with precipitates ................................. 113

Perform the prerun checklist ............................................... 114

Run the prepare GeneTitan™ reagent trays step .............................. 115

Stage 5 and stage 6 for the 8-plate workﬂow ................................. 121

Workﬂow for Stage 6: Prepare GeneTitan™ reagent trays ...................... 123

■CHAPTER 5 Process array plates with the GeneTitan™ Multi-Channel

(MC) Instrument .................................................................. 130

Stage 1: Create and upload a GeneTitan™ Array Plate Registration ﬁle ............... 131

Stage 2: Hybridize plates in the GeneTitan™ MC Instrument ........................ 133

Materials, labware, and reagents required .................................... 133

Set up the instrument ..................................................... 134

Load an array plate and hybridization tray into the GeneTitan™ MC

Instrument (for Hyb-Wash-Scan or Hyb-Wash) ............................. 138

Load a second array plate and hybridization tray onto the GeneTitan™

MC Instrument ......................................................... 141

Queue a second plate for scanning ......................................... 143

Stage 3: Ligate, wash, stain, and scan ........................................... 145

The GeneTitan™ tray loading process ....................................... 145

Load trays in GeneTitan™ MC Instrument .................................... 145

Continue the scan workﬂow .................................................... 151

Shut down the GeneTitan™ MC Instrument ....................................... 152

■CHAPTER 6 8-plate workﬂow for PharmacoScan™ Array Plates ............ 153

Overview of the 8-plate workﬂow ............................................... 154

Overview of the manual mPCR and automated target preparation steps ......... 156

Time required for assay steps .............................................. 157

Thaw frozen plates of ampliﬁed DNA ........................................ 159

Thaw plates with frozen pellets ............................................. 160

Target preparation and array processing for the 8-plate workﬂow ................... 160

Day 1 activities ........................................................... 160

Day 2 activities ........................................................... 164

Day 3 activities ........................................................... 168

Day 4 activities ........................................................... 173

Day 5 activities ........................................................... 177

Contents

6PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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■CHAPTER 7 3-plate workﬂow for PharmacoScan™ Array Plates using a

3-hour DNA precipitation step .................................................. 181

Overview of the 3-plate workﬂow with 3-hour precipitation ......................... 182

Time required for assay steps .............................................. 185

Thaw the frozen plate of ampliﬁed DNA ...................................... 187

Target preparation and array processing for the 3-plate workﬂow using a 3-hour

precipitation step ........................................................... 187

Day 1 activities ........................................................... 188

Day 2 activities ........................................................... 191

Day 3 activities ........................................................... 195

Day 4 activities ........................................................... 199

■APPENDIX A Recommended techniques for GeneTitan™ MC

Instrument operation ............................................................ 201

Array plate packaging ......................................................... 202

Proper tray alignment and placement ........................................... 202

Scan tray ................................................................ 204

Proper orientation of consumables .......................................... 205

Drawer tabs in the GeneTitan™ MC Instrument ............................... 206

Stain trays and covers ......................................................... 207

Label GeneTitan™ hybridization and reagent trays ................................. 207

Label the GeneTitan™ 96-layout hybridization tray ............................ 207

Label the GeneTitan™ reagent trays ......................................... 208

Deionization of GeneTitan™ trays and covers ..................................... 208

Deionize GeneTitan™ trays and covers ...................................... 210

Ion-indicator cap ......................................................... 211

Setup options for array plate processing ......................................... 211

Hyb-Wash-Scan .......................................................... 212

Hyb-Wash ............................................................... 212

Wash-Scan .............................................................. 213

Wash-Scan Resume ...................................................... 213

Scan .................................................................... 214

Unload Plates ............................................................ 214

When to abort a process ....................................................... 214

Abort a process .......................................................... 215

Email notiﬁcations from the GeneTitan™ MC Instrument ............................ 216

GeneTitan™ MC Instrument lamp ............................................... 216

■APPENDIX B Register samples in GeneChip™ Command Console™....... 218

GeneTitan™ Array Plate Registration ﬁle .......................................... 218

Create a GeneTitan™ Array Plate Registration ﬁle .................................. 218

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■APPENDIX C Fragmentation quality control gel protocol .................... 221

Equipment required ........................................................... 221

E‑Gel™ and reagents required .................................................. 221

Consumables required ......................................................... 222

Prepare the gel diluent ......................................................... 222

Dilute the TrackIt™ Cyan/Orange Loading Buer .............................. 222

Dilute the 25 bp DNA ladder ............................................... 222

Run the fragmentation QC gel .................................................. 222

■APPENDIX D Sample quantiﬁcation after resuspension ..................... 224

Equipment required ........................................................... 224

Spectrophotometer ....................................................... 224

Quantify the diluted samples ................................................... 224

OD yield evaluation guidelines .................................................. 225

Plate reader guidelines for sample quantiﬁcation .................................. 225

■APPENDIX E mPCR quality control gel protocol .............................. 226

Materials required ............................................................. 226

Prepare the 50 bp DNA Ladder ................................................. 227

Prepare mPCR samples for gel analysis .......................................... 227

Run the mPCR QC gel ......................................................... 228

■APPENDIX F Troubleshooting ................................................... 230

GeneTitan™ MC Instrument support ﬁles for troubleshooting ....................... 230

Log ﬁles ................................................................. 230

GeneChip™ Command Console™ log ﬁles .................................... 230

Other GeneChip™ Command Console™ ﬁles ................................. 230

GCC log ﬁles for GeneTitan™ MC Instrument systems ......................... 231

Troubleshooting the GeneTitan™ MC Instrument .................................. 232

GeneTitan™ MC Instrument ﬂuidic diagnostic messages ........................... 236

■APPENDIX G GeneTitan™ Multi-Channel Instrument care ................... 239

Overview .................................................................... 239

Maintenance ................................................................. 239

Monthly ................................................................. 239

Every 6 months ........................................................... 240

Outer enclosure fan ﬁlters ...................................................... 240

Cleaning schedule ........................................................ 240

Clean the GeneTitan™ MC Instrument fan ﬁlter ............................... 240

Contents

8PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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Bottle ﬁlter replacement ....................................................... 241

Remove and inspect the reagent bottle ﬁlters ................................ 241

Replace ﬂuidics bottle ﬁlter ................................................ 242

Xenon lamp replacement in the GeneTitan™ MC Instrument ........................ 242

Lamp life/imaging device status notices ..................................... 242

Remove the xenon lamp ................................................... 244

Replace the xenon lamp ................................................... 245

Reset the lamp life counter ................................................ 246

■APPENDIX H Routine care for the Applied Biosystems™ NIMBUS™

Target Preparation Instrument .................................................. 247

O-ring care ................................................................... 247

Tip isolator ................................................................... 248

Clean the tip isolator ...................................................... 249

Trash chute .................................................................. 250

Assemble the trash chute .................................................. 250

Trash chute cleaning ...................................................... 253

Thermoshake device maintenance .............................................. 254

■APPENDIX I Safety ............................................................... 255

Symbols on this instrument .................................................... 255

Standard safety symbols .................................................. 255

Location of safety labels ................................................... 257

Control and connection symbols ........................................... 258

Conformity symbols ...................................................... 259

Safety information for instruments not manufactured by Thermo Fisher Scientiﬁc ..... 260

Instrument safety ............................................................. 260

General ................................................................. 260

Physical injury ............................................................ 261

Electrical safety .......................................................... 262

Cleaning and decontamination ............................................. 263

Instrument component and accessory disposal .............................. 263

Safety and electromagnetic compatibility (EMC) standards ......................... 264

Safety standards ......................................................... 264

EMC standards ........................................................... 264

Environmental design standards ............................................ 265

Chemical safety .............................................................. 266

Biological hazard safety ....................................................... 268

Contents

PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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■Documentation and support ...................................................... 269

Related documentation ........................................................ 269

Customer and technical support ................................................ 270

Limited product warranty ...................................................... 271

Contents

10 PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

Preparation Instrument

Overview

■Introduction .......................................................................... 11

■About the PharmacoScan™ Assay 96-Array Format Automated Workﬂow ................... 12

■Multi-plate workﬂows ................................................................. 13

■Overview of the PharmacoScan™ Assay 96-Array Format Automated Workﬂow .............. 14

Developed in collaboration with experts across the ﬁeld of Pharmacogenomics, PharmacoScan™

Solution is the industry’s broadest content genetic analysis system speciﬁcally designed to provide

insight into the absorption, distribution, metabolism, and excretion (ADME) and transport of commonly

prescribed medicines. By interrogating more than 4,600 markers in almost 1,200 genes known to play

a role in drug metabolism, traditional clinical researchers gain unprecedented understanding into an

individual’s ability to process those drugs with high evidence for genetic association, as well as those

markers where moderate, low, preliminary, and unknown evidence exists.

PharmacoScan™ Solution uses the same chemistry from the Axiom™ 2.0 Assay and GeneTitan™ Multi-

Channel Instrument, a system that is preferred worldwide by genetic researchers for the ecient

workﬂow, high-throughput, economic pricing, and reproducibility of results critical to multi-year data

collection and analysis eorts.

Introduction

PharmacoScan™ Assay 96-Array Format Automated Workﬂow is available as a bundled kit that includes

the arrays, reagents, and consumables needed for processing one 96-format plate, each having 94

samples and 2 controls. The automated workﬂow described in this user guide uses the Axiom™

96 Sample Automated Target Preparation Solution method Applied Biosystems™ NIMBUS™ Target

Preparation Instrument and GeneTitan™ reagent preparation. The multiplex PCR (mPCR) and mPCR

spike-in steps are not part of the automated method and must be executed o-deck, manually.

The PharmacoScan™ Assay interrogates biallelic and multiallelic SNPs, indels, and copy number

variation (CNV) in a single assay workﬂow. Starting with genomic DNA, the samples are processed

by performing an automated target preparation protocol followed by automated processing of the array

plates on the GeneTitan™ Multi-Channel (MC) Instrument.

•Target preparation uses methods including DNA ampliﬁcation, fragmentation, precipitation, and

resuspension of the target in hybridization cocktail.

•The hybridization-ready targets are then transferred to the GeneTitan™ MC Instrument for

automated, hands-free processing including hybridization, staining, washing, and scanning.

PharmacoScan™ Solution provides pharmacogenomic-variation information for greater than 4,600

ADME markers in almost 1,200 genes. This content is sourced from globally endorsed consortium

databases including, but not limited to CPIC, PharmGKB, and PharmaADME. Also included on

PharmacoScan™ Array Plates are high value markers for human leukocyte antigen (HLA) imputation,

1

PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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markers for killer cell immunoglobulin-like receptors (KIR), markers for human ancestry identiﬁcation

(AIM), a marker GWAS backbone, and markers for sample ID and tracking. The combination of these

high value markers, in addition to PharmacoScan’s ability to precisely call variants in critical genes on

a microarray, compliments Thermo Fisher Scientiﬁc’s current solutions for Pharmacogenomics using

OpenArray™ real-time PCR, digital PCR, and Ion AmpliSeq™ NGS panels.

The PharmacoScan™ Assay is a multiplex genotyping assay which combines the proven Axiom™

chemistry with the incorporation of a multiplex PCR step to overcome some complexities associated

with genotyping highly homologous markers. PharmacoScan™ software and algorithm developments

include an allele translation and phenotyping tool and copy number aware genotyping. Array plates are

processed on a GeneTitan™ MC Instrument controlled by Applied Biosystems™ GeneChip™ Command

Console™ 4.3 or later. The resulting CEL ﬁles are analyzed by Axiom™ Analysis Suite 3.1 or later, or by

Applied Biosystems™ Array Power Tools 1.19 or later.

About the PharmacoScan™ Assay 96-Array Format

Automated Workﬂow

The PharmacoScan™ Assay 96-Array Format Automated Workﬂow is a fully automated workﬂow for

high-throughput that requires the following sets of steps:

1. Genomic DNA preparation—Resulting in samples that meet requirements detailed in Chapter 2,

“Prepare genomic DNA”.

2. A manual preparation of a multiplex PCR step (mPCR) followed by automated target preparation of

the samples (see Chapter 4, “Multiplex PCR and target preparation with the Applied Biosystems™

NIMBUS™ Target Preparation Instrument”).

3. Target preparation of the samples: Chapter 4, “Multiplex PCR and target preparation with the

Applied Biosystems™ NIMBUS™ Target Preparation Instrument”

4. Array processing, done with:

•GeneTitan™ MC Instrument

•GeneTitan™ Instrument Control software

•Applied Biosystems™ GeneChip™ Command Console™ Portal software

See Chapter 5, “Process array plates with the GeneTitan™ Multi-Channel (MC) Instrument”.

A list of the required equipment and supplies for running the PharmacoScan™ Assay 96-Array Format

Automated Workﬂow on the Applied Biosystems™ NIMBUS™ Target Preparation Instrument can be

found in the PharmacoScan™ Assay 96-Array Format Automated Workﬂow Site Preparation Guide—

Applied Biosystems™ NIMBUS™ Target Preparation Instrument, Pub. No. MAN0018102.

Chapter 1 Overview

About the PharmacoScan™ Assay 96-Array Format Automated Workﬂow

1

12 PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

Preparation Instrument

Assay highlights

This user guide introduces an option for a 3-hour DNA target precipitation step to enable a faster assay

turnaround time, going from sample to CEL ﬁle generation within 72 hours. The 3-hour precipitation

step shortens “Stage 2: Fragment and precipitate the DNA” to enable the operator to advance to

“Stage 3: Centrifuge and dry pellets”, followed by “Stage 4A and 4B: Resuspension and hybridization

preparation”, “Stage 4C: Perform sample QC”, and the start of “Stage 5: Prepare the hybridization tray”

on page 102 to start hybridization on the GeneTitan™ MC Instrument on day 2 of the assay workﬂow.

Note that this workﬂow option requires approximately 9–10 hours to complete the combined day 2

activities (fragmentation to initiation of hybridization on the GeneTitan™ MC Instrument).

In addition, a recommended workﬂow is presented to support the processing of 3 plates per week

using the shortened DNA precipitation step. See Chapter 7, “3-plate workﬂow for PharmacoScan™

Array Plates using a 3-hour DNA precipitation step” for details regarding equipment and operator

assumptions to support this workﬂow.

The standard PharmacoScan™ Assay workﬂow, in which the DNA is precipitated overnight, provides

a convenient stopping point to support single operator assay execution of 1 plate within an 8-hour

workday. Recommended workﬂows are presented to support the processing of 3 plates per week

using 3-hour DNA precipitation, and 8 plates per week using the standard overnight DNA precipitation.

See the appropriate chapter for details regarding equipment and operator assumptions to support

these workﬂows. (Chapter 7, “3-plate workﬂow for PharmacoScan™ Array Plates using a 3-hour DNA

precipitation step”, and Chapter 6, “8-plate workﬂow for PharmacoScan™ Array Plates”.)

Multi-plate workﬂows

Thermo Fisher Scientiﬁc supports high-throughput workﬂows that allow you to run a set of samples and

array plates through the protocol using a minimum number of personnel and an extended week. The

timing of steps is critical because of the following limits:

•Incubation for DNA ampliﬁcation is 22–24 hours.

•Hybridization in the GeneTitan™ MC Instrument is 23.5–24 hours.

•Reagent trays for wash/stain/imaging must be prepared as hybridization ﬁnishes.

•Limits to when a second hybridization tray and array plate can be loaded into the GeneTitan™ MC

Instrument.

Using the automated target preparation protocol, workﬂows are available for processing 8 array plates

per work week, or 3 array plates per week.

•Chapter 6, “8-plate workﬂow for PharmacoScan™ Array Plates”

•Chapter 7, “3-plate workﬂow for PharmacoScan™ Array Plates using a 3-hour DNA precipitation

step”

Chapter 1 Overview

Multi-plate workﬂows 1

PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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Overview of the PharmacoScan™ Assay 96-Array Format

Automated Workﬂow

gDNA plate setup 5 ng/μL of gDNA

Day 1 ▼ ▼Manual procedure

10 µL/well 20 µL/well 10 µL/well from the gDNA plate

is used for Stage 1A, and

20 µL/well from the gDNA plate

is used for Stage 1B.

Day

1

Stage 1A: mPCR Stage 1B: DNA ampliﬁcation

(manual) (automated)

23 ±1 hour ampliﬁcation

incubation at 37°C.

Optional stopping point.

▼ ▼

Day 2 mPCR Plate ► Ampliﬁcation Plate

▼

10 µL mPCR spike-in to

Ampliﬁcation Plate

Day 2 “Stage 2: Fragment and precipitate the DNA” on page 61

▼

Approximate time: 16-24 hours

(or optional 3-hour precipitation

step) –20°C incubation of

Precipitation Plate

Day 3 “Stage 3: Centrifuge and dry pellets” on page 75

▼Optional stopping point.

Day 3 “Stage 4A: Prepare the resuspension buer” on page 80

“Stage 4B: Prepare Hybridization Master Mix” on page 87

“Stage 4C: Perform sample QC” on page 94

▼

Day 3 “Stage 5: Prepare the hybridization tray” on page 102

▼

23.5 to 24-hour array

hybridization in the GeneTitan™

MC Instrument.

Day 4 “Stage 6: Prepare GeneTitan™ reagent trays” on page 110

Array processing

Day 5 Process array plates with the GeneTitan™ Multi-Channel (MC)

Instrument (page 130)

Array processing is completed with the GeneTitan™ MC Instrument and

GeneChip™ Command Console™ software v4.3 or later.

Fluidics: 5 hours

Scan: ~7.5 hours

Chapter 1 Overview

Overview of the PharmacoScan™ Assay 96-Array Format Automated Workﬂow

1

14 PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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Prepare genomic DNA

■Sources of genomic DNA .............................................................. 15

■General requirements ................................................................. 16

■Genomic DNA extraction and puriﬁcation methods ....................................... 18

■Clean up genomic DNA ............................................................... 19

■Genomic DNA preparation ............................................................. 19

■GeneTitan™ Array Plate Registration ﬁle ................................................. 23

The general requirements for genomic DNA (gDNA) sources and extraction methods are described

in this chapter. The success of this assay requires uniform ampliﬁcation of the genome starting with

relatively intact gDNA. To achieve uniform ampliﬁcation, the gDNA must be of high quality, and must be

free of contaminants that can aect the enzymatic reactions to be performed.

The PharmacoScan™ Reagent Kit 96 Reactions (Cat. No. 913025) is used for this protocol. The kit

contains 2 control gDNAs: Control DNA 1 and Control DNA 2. This DNA meets the requirements

outlined below, and both control DNAs must be included on every plate for data analysis purposes. The

size and purity of sample gDNA can be compared with those of the control DNA to evaluate sample

quality.

Assay performance can vary for gDNA samples that do not meet the general requirements. However,

the reliability of any given result must be evaluated in the context of overall experimental design and

goals.

Sources of genomic DNA

The following sources of human gDNA have been successfully tested in the laboratories at Thermo

Fisher Scientiﬁc for DNA that meets the requirements for the PharmacoScan™ Assay.

•Whole blood

•Buccal cell

•Saliva

•Cell lines

Other sample types have not been veriﬁed in this assay and are not currently supported.

Note: DNA derived from formalin‑ﬁxed paran-embedded (FFPE) blocks must not be used with this

assay.

2

PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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General requirements

•Starting DNA must be double-stranded for accurate concentration determination.

•DNA must be of high purity. DNA must be free of DNA polymerase inhibitors. Examples of inhibitors

include high concentrations of heme (from blood) and high concentrations of chelating agents (that

is, EDTA). The gDNA extraction and puriﬁcation method must create DNA that is salt-free because

high concentrations of particular salts can also inhibit enzyme reactions. DNA purity indicated by

OD260/OD280 and OD260/OD230 ratios. The OD260/OD280 ratio should be between 1.8 and 2.0 and

the OD260/OD230 ratio should be greater than 1.5. We recommend that DNA samples that do not

meet these criteria be cleaned up as described in “Clean up genomic DNA” on page 19.

•DNA must not be degraded. The average size of gDNA can be evaluated on a 1% agarose gel

using an appropriate size standard control. Approximately 90% of the DNA must be greater than

10 Kb in size. Control DNA can be run on the same gel for comparison.

Note: DNA size integrity is important for successful assay performance. It is strongly advised to

evaluate gDNA by gel electrophoresis. Evaluating the gDNA by gel electrophoresis is of particular

importance for DNA extracted from saliva and buccal cells, sample types prone to DNA degradation.

Special requirements

Preampliﬁcation area

Precautions are required when manipulating genomic DNA to avoid contamination with foreign DNA

ampliﬁed in other reactions and procedures. It is recommended that genomic DNA manipulations are

performed in a dedicated preampliﬁcation room or area separate from the main laboratory.

This preampliﬁcation area requires a dedicated set of pipettes and plasticware. If no dedicated area is

available, use of a dedicated bench or a dedicated biosafety hood and dedicated pipettes is suggested.

If no dedicated bench or biosafety hood is available, a set of dedicated pipettes is recommended.

Ideally, this preampliﬁcation area would be separate from the ampliﬁcation staging area, however, these

areas may be combined due to space and equipment limitations.

Chapter 2 Prepare genomic DNA

General requirements

2

16 PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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Evaluate the quality of genomic DNA with 1% agarose E‑Gel™

We recommend this quality control step to evaluate the quality of the gDNA before starting the assay.

Equipment and reagents required

Unless otherwise indicated, all materials are available through thermoﬁsher.com.

Item Source

Invitrogen™ Mother E-Base™ Device EBM03

Invitrogen™ Daughter E-Base™ Device (optional for running multiple gels in parallel) EBD03

Invitrogen™ E‑Gel™ 48 Agarose Gels, 1% G800801

Invitrogen™ RediLoad™ Loading Buer 750026

Invitrogen™ E‑Gel™ 96 High Range DNA Marker 12352019

Guidelines for preparing the gDNA Sample Plate for gel analysis

The following guidelines are recommended when preparing the gDNA Sample Plate for gel analysis.

•Load a DNA mass of 10 ng to 20 ng per well (recommended). If lower amounts are loaded,

omission of the loading dye is recommended to improve visualization. Loading ≥25-ng gDNA per

well can improve the image.

•Add 3 µL of 0.1X of RediLoad™ Loading Buer (RediLoad™ Loading Buer dye diluted 10-fold with

nuclease-free water) dye to each sample.

•Bring each sample to a total volume of 20 µL using nuclease-free water. For example, if the volume

of genomic DNA is 5 µL, add 3 µL of RediLoad™ Loading Buer, then bring to 20 µL total by adding

12 µL of water.

•Seal, vortex, and centrifuge brieﬂy.

Run a 48-lane 1% agarose E‑Gel™

1. Power on the E-Base™ device (red light).

2. Push Power/Prg to ensure that the gel base is in EG mode, not EP mode.

3. Insert the E‑Gel™ 48 Agarose Gels, 1% into the slot.

4. Remove 2 combs.

5. Load 20 µL of gDNA samples onto the E‑Gel™ 48 Agarose Gels, 1%.

6. If needed, load 15 µL of diluted E‑Gel™ 96 High Range DNA Marker (1:3 dilution or ~0.34X from

stock) into all marker wells.

7. Fill all empty wells with water.

Chapter 2 Prepare genomic DNA

General requirements 2

PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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17

8. Adjust the run time to ~27 minutes.

9. Push Power/Prg again.

It changes from red to green.

When run time is reached (the ladder band reaches the end of the lane), the system automatically shuts

o. The gel is ready for imaging.

E‑Gel™ results

The following ﬁgure shows gel images of intact gDNA (that is appropriate for use in the PharmacoScan™

Assay) and degraded gDNA samples. For gDNA that is degraded perform a test experiment to

investigate the performance of the samples in the PharmacoScan™ Assay before starting any large-

scale genotyping projects.

10 kb

2 kb

4 kb

0.8 kb

0.4 kb

1 2

Figure 1 Gel images with intact gDNA and degraded gDNA.

1Intact samples 2Degraded samples

Genomic DNA extraction and puriﬁcation methods

Genomic DNA extraction and puriﬁcation methods that meet the general requirements that are outlined

are expected to yield successful results. Methods that include boiling or strong denaturants are

not acceptable because the DNA would be made single-stranded and can no longer be accurately

quantiﬁed using a PicoGreen™-based assay.

Chapter 2 Prepare genomic DNA

Genomic DNA extraction and puriﬁcation methods

2

18 PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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Clean up genomic DNA

If a gDNA preparation is suspected to contain inhibitors, the following cleanup procedure can be used.

1. Add 0.5 volumes of 7.5 M NH4OAc, 2.5 volumes of absolute ethanol (stored at −20°C), to gDNA.

2. Vortex, then incubate at −20°C for 1 hour.

3. Centrifuge at 12,000 × g in a microcentrifuge at room temperature for 20 minutes.

4. Remove supernatant, then wash pellet with 80% ethanol.

5. Centrifuge at 12,000 × g at room temperature for 5 minutes.

6. Remove the 80% ethanol, then repeat the 80% ethanol wash 1 more time.

7. Resuspend the pellet in Reduced EDTA TE Buer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).

Genomic DNA preparation

This step must be done before proceeding with the mPCR and DNA ampliﬁcation stages.

The genomic DNA (gDNA) you process using the PharmacoScan™ Assay must meet the general

requirements that are listed earlier in this chapter. The amount of gDNA is 50 ng for the mPCR step and

100 ng for the PharmacoScan™ whole-genome ampliﬁcation step.

Genomic DNA input requirements

Sample type Volume per well Input mass

per well

gDNA

concentration

Stage 1A: mPCR 10 µL 50 ng 5 ng/μL

Stage 1B: DNA ampliﬁcation 20 µL 100 ng 5 ng/μL

Time required

Allow 30–60 minutes for reagents to thaw and 30 minutes for setup.

Chapter 2 Prepare genomic DNA

Clean up genomic DNA 2

PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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19

Equipment, consumables, and reagents required

Unless otherwise indicated, all materials are available through thermoﬁsher.com.

Equipment and consumables required

Quantity Item

As required Adhesive seals for plates

1 Ice bucket, ﬁlled with ice

1 each Pipettes: single-channel P10 or P20

Optional: multichannel P10 or P20

As required Pipette tips

1 Round deepwell plate, one of the following:

•Axygen™ 96-well Round Deepwell Plate (sterile, PDW20CS), Fisher Scientiﬁc™,

14-222-354.

•Axygen™ 96-well Round Deepwell Plate (nonsterile, PDW20C), Fisher Scientiﬁc™,

14-222-353.

•PerkinElmer™ Deepwell StorPlate-96 U Round, 2.0mL (6008490 only), Fisher

Scientiﬁc™, 50-905-1741.

1 96‑well PCR plate, one of the following:

•Bio-Rad™ HSS9641 (green) or HSS9601 (clear) for Applied Biosystems™

GeneAmp™ PCR System 9700, Applied Biosystems™ Veriti™ Thermal Cycler, and

Applied Biosystems™ ProFlex™ PCR System.

•Bio-Rad™ HSP9631 (blue) or HSP9601 (white) for the Eppendorf™ Mastercycler™

pro S.

1 Plate centrifuge

1 Plate spectrophotometer (required only if no OD measurements available for samples)

1 Vortexer

Chapter 2 Prepare genomic DNA

Genomic DNA preparation

2

20 PharmacoScan™ Assay 96-Array Format Automated Workﬂow User Guide—Applied Biosystems™ NIMBUS™ Target

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Thermo Fisher Scientific PharmacoScan Assay 96-Array Format Automated Workflow User guide

Thermo Fisher Scientific PharmacoScan Assay 96-Array Format Automated Workflow User guide

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