Illumina Two-Sample HC Infinium II Assay User Guide

Infinium

®

II Assay

Two-Sample HC Protocols

FOR RESEARCH ONLY

ILLUMINA PROPRIETARY

Part # 11280749 Rev. A

Notice

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OPERATING THE SAME.

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Infinium II Assay Two-Sample HC Protocols iii

Revision History

Part Number and Revision

11230151, Rev. A

11280749, Rev. A

Date

December 2006

October 2007

Infinium II Assay Two-Sample HC Protocols v

Table of Contents

Notice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

Revision History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Table of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Chapter 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Audience and Purpose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

The Infinium II Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

The Two-Sample BeadChip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Illumina Lab Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Tracking Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Tecan GenePaint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Illumina BeadArray Reader. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Illumina BeadStudio Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Technical Assistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Chapter 2 Two-Sample HC BeadChip Manual Protocol . . . . . . . . . . . 13

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Two-Sample BeadChip Manual Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Equipment, Materials, and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Make Standard DNA Plate (Optional/Infinium LIMS) . . . . . . . . . . . . . . . . . . 17

Make Quant (Optional/LIMS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Read Quant (Optional/LIMS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Prepare the WG#-DNA Plate (Optional) . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Make the AMP2 Plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Incubate the AMP2 Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Fragment the AMP2 Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Precipitate the AMP2 Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Resuspend the AMP2 Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Hybridize HC BC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Wash BC2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Single-Base Extension and Stain BC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Image BC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Infinium II Assay Two-Sample HC Protocols vii

viii Table of Contents

Chapter 3 Two-Sample HC BeadChip Automated Protocol . . . . . . . . 73

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Equipment, Materials, and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Make Standard DNA Plate (Optional/Infinium LIMS) . . . . . . . . . . . . . . . . . . 78

Make Quant (Optional/LIMS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Read Quant (Optional/LIMS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Make the AMP2 Plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Make Multiple AMP2 Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

Incubate the AMP2 Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

Fragment the AMP2 Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Precipitate the AMP2 Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Resuspend the AMP2 Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Hybridize HC BC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

Wash BC2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Single-Base Extension & Stain BC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Image BC2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Part # 11280749 Rev. A

List of Tables

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Sample Sheet Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Illumina Customer Support Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Illumina-Supplied Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

User-Supplied Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Illumina-Supplied Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Concentration of Lambda DNA Standards . . . . . . . . . . . . . . . . . . . . . . . . . 19

PicoGreen Reagent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

User-Supplied Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Illumina-Supplied Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

User-Supplied Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Illumina-Supplied Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Concentration of Lambda DNA Standards . . . . . . . . . . . . . . . . . . . . . . . . . 80

PicoGreen Reagent Volumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

List of Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

Infinium II Assay Two-Sample HC Protocols ix

x List of Tables

Part # 11280749 Rev. A

List of Figures

Chapter 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

Figure 1

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Denaturing and Neutralizing DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Incubating DNA to Amplify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Fragmenting DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Precipitating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Resuspending DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Hybridizing DNA to BeadChip. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Washing BeadChip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Extending and Staining BeadChip. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Imaging BeadChip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Sample Sheet Example . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Chapter 2 Two-Sample HC BeadChip Manual Protocol . . . . . . . . . . . 13

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Two-Sample BeadChip Manual Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . 15

MIDI Plate Wells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Standard QNT Plate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

QNT Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Infinium Fluorometry Analysis Opening Screen. . . . . . . . . . . . . . . . . . . . . . 23

Number of QNT Plates Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Plate Barcode Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Fluorescence Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Distributing Sample to WG#-DNA Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Denaturing and Neutralizing DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Distributing Sample to AMP2 Plate Wells . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Incubating DNA to Amplify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Fragmenting DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Distributing Sample in AMP2 Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Securing Plates to Vortexer Platform with Velcro Straps . . . . . . . . . . . . . . . 30

Precipitating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Balancing AMP2 Plate in Centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

Uncovered AMP2 Plate Inverted for Air Drying . . . . . . . . . . . . . . . . . . . . . . 34

Resuspending DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Hybridizing DNA to BeadChip. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

BeadChip Hyb Cartridge Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Hyb Chamber and Gasket . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Placing Gasket into Hyb Chamber. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Hyb Chamber with Gasket in Place . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Infinium II Assay Two-Sample HC Protocols xi

xii List of Figures

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Dispensing PB2 into Hyb Chamber Reservoirs . . . . . . . . . . . . . . . . . . . . . . 40

Sealing the Hyb Chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Consolidating Sample Back Into Original Sample Well. . . . . . . . . . . . . . . . 42

Placing BeadChips into Hyb Chamber Inserts . . . . . . . . . . . . . . . . . . . . . . . 42

Distributing Sample to BeadChips (Pattern) . . . . . . . . . . . . . . . . . . . . . . . . 43

Dispensing Sample onto BeadChip (Photo) . . . . . . . . . . . . . . . . . . . . . . . . 44

Placing Hyb Chamber Inserts into Hyb Chamber . . . . . . . . . . . . . . . . . . . . 44

Securing Hyb Chamber Lid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Hyb Chamber Correctly Placed in Hyb Oven . . . . . . . . . . . . . . . . . . . . . . . 45

Two Hyb Chambers Correctly Placed in Hyb Oven. . . . . . . . . . . . . . . . . . . 46

Incorrectly Placed Hyb Chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Washing BeadChip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Wash Rack in Wash Dish Containing WB1. . . . . . . . . . . . . . . . . . . . . . . . . . 51

Removing the Coverseal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Placing BeadChips in Wash Dish Containing WB1 . . . . . . . . . . . . . . . . . . . 52

Placing Black Frames into Alignment Fixture . . . . . . . . . . . . . . . . . . . . . . . 53

Placing BeadChip into Black Frame on Alignment Fixture . . . . . . . . . . . . . 53

Placing Clear Plastic Spacer onto BeadChip . . . . . . . . . . . . . . . . . . . . . . . . 54

Placing Alignment Bar onto Alignment Fixture . . . . . . . . . . . . . . . . . . . . . . 54

Placing Glass Back Plate onto BeadChip . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Securing Flow-Through Chamber Assembly with Metal Clamps. . . . . . . . . 56

Trimming Spacer Ends from Flow-Through Chamber Assembly . . . . . . . . . 56

Extending and Staining BeadChip. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

XStain BC2 Reagent Tubes and Bottles. . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Water Circulator Connected to Chamber Rack . . . . . . . . . . . . . . . . . . . . . . 60

Illumina Temperature Probe in Chamber Rack . . . . . . . . . . . . . . . . . . . . . . 61

Dispensing RA1 into Each Flow-Through Chamber . . . . . . . . . . . . . . . . . . 62

PB1 and XC4 Wash Dishes with Staining Rack . . . . . . . . . . . . . . . . . . . . . . 64

Staining Rack Locking Arms and Tabs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Removing Metal Clamps from Flow-Through Chamber . . . . . . . . . . . . . . . 65

Washing BeadChips in PB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Moving BeadChips from PB1 to XC4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Staining Rack in Correct Orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Moving the Staining Rack from XC4 to Tube Rack . . . . . . . . . . . . . . . . . . . 68

Removing Staining Rack Handle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Placing BeadChips on Tube Rack . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Testing Vacuum Seal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

Chapter 3 Two-Sample HC BeadChip Automated Protocol . . . . . . . . 73

Figure 72

Figure 73

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Figure 83

Two-Sample HC BeadChip Automated Workflow . . . . . . . . . . . . . . . . . . . . 75

MIDI Plate Wells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Tecan Eight-Tip Robot (Make Quant Setup) . . . . . . . . . . . . . . . . . . . . . . . . 82

Make Quant Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Infinium Fluorometry Analysis Opening Screen. . . . . . . . . . . . . . . . . . . . . . 85

Number of QNT Plates Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Plate Barcode Dialog Box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Fluorescence Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Denaturing and Neutralizing DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Tecan Eight-Tip Robot (Make AMP2 Setup) . . . . . . . . . . . . . . . . . . . . . . . . 89

Selecting the DNA Plate Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

Make AMP2 Basic Run Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Part # 11280749 Rev. A

List of Figures xiii

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Securing Plates to Vortexer Platform with Velcro Straps . . . . . . . . . . . . . . . 91

Selecting Project or Batch for Make AMP2 . . . . . . . . . . . . . . . . . . . . . . . . . 92

Make AMP2 Screen with Project and Batch Selected . . . . . . . . . . . . . . . . . 92

Tecan Eight-Tip Robot (Make Multi AMP2 Setup) . . . . . . . . . . . . . . . . . . . . 95

Selecting Project for Make Multi AMP2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Selecting Batches. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Make Multi AMP2 Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

Incubating DNA to Amplify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

(Infinium LIMS) Verifying AMP2 for Incubation . . . . . . . . . . . . . . . . . . . . . 100

Fragmenting DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Tecan Eight-Tip Robot (Fragment AMP2 Setup) . . . . . . . . . . . . . . . . . . . . 102

Fragment AMP2 Screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Precipitating DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

Tecan Eight-Tip Robot (Precip AMP2 Setup) . . . . . . . . . . . . . . . . . . . . . . . 105

(Infinium LIMS) Verifying AMP2 for Centrifugation . . . . . . . . . . . . . . . . . . 106

Precip AMP2 Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Balancing AMP2 Plate in Centrifuge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

Uncovered AMP2 Plate Inverted for Air Drying . . . . . . . . . . . . . . . . . . . . . 110

Resuspending DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

Tecan Eight-Tip Robot (Resuspend AMP2 Setup) . . . . . . . . . . . . . . . . . . . 112

Resuspend AMP2 Screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Hybridizing DNA to BeadChip. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

BeadChip Hyb Cartridge Components . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Hyb Chamber and Gasket . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Placing Gasket into Hyb Chamber. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

Hyb Chamber with Gasket in Place . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118

Dispensing PB2 into Hyb Chamber Reservoirs . . . . . . . . . . . . . . . . . . . . . 118

Sealing the Hyb Chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Placing Alignment Fixtures and AMP2 Plate onto Robot Bed . . . . . . . . . . 120

(Infinium LIMS) Verifying AMP2 and BeadChips for Hyb . . . . . . . . . . . . . . 121

Placing BeadChips into Robot Alignment Fixture . . . . . . . . . . . . . . . . . . . 122

Four Stacked Robot Alignment Fixtures . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Hyb HC BC2 Screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

Placing BeadChips into Hyb Chamber Inserts . . . . . . . . . . . . . . . . . . . . . . 125

Placing Hyb Chamber Inserts into Hyb Chamber . . . . . . . . . . . . . . . . . . . 125

Securing Hyb Chamber Lid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Hyb Chamber Correctly Placed in Hyb Oven . . . . . . . . . . . . . . . . . . . . . . 126

Two Hyb Chambers Correctly Placed in Hyb Oven. . . . . . . . . . . . . . . . . . 127

Incorrectly Placed Hyb Chamber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Washing BeadChip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

(Infinium LIMS) Verifying Reagents and BeadChips for Washing. . . . . . . . 131

Wash Rack in WB1 Wash Dish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Removing the Coverseal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Placing BeadChips in WB1 Wash Dish. . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Placing Black Frames into Alignment Fixture . . . . . . . . . . . . . . . . . . . . . . 134

Placing BeadChip into Black Frame on Alignment Fixture . . . . . . . . . . . . 134

Placing Clear Plastic Spacer onto BeadChip . . . . . . . . . . . . . . . . . . . . . . . 135

Placing Alignment Bar onto Alignment Fixture . . . . . . . . . . . . . . . . . . . . . 135

Placing Glass Back Plate onto BeadChip . . . . . . . . . . . . . . . . . . . . . . . . . . 136

Securing Flow-Through Chamber Assembly with Metal Clamps. . . . . . . . 137

Trimming Spacer Ends from Flow-Through Chamber Assembly . . . . . . . . 137

Extending and Staining BeadChip. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Infinium II Assay Two-Sample HC Protocols

xiv List of Figures

Figure 136

Figure 137

Figure 138

Figure 139

Figure 140

Figure 141

Figure 142

Figure 143

Figure 144

Figure 145

Figure 146

Figure 147

Figure 148

Figure 149

Figure 150

Figure 151

Figure 152

Figure 153

Figure 154

Figure 155

Figure 156

Figure 157

Figure 158

Figure 159

Figure 160

Figure 161

Figure 162

Figure 163

Water Circulator Connected to Chamber Rack . . . . . . . . . . . . . . . . . . . . . 140

Illumina Temperature Probe in Chamber Rack . . . . . . . . . . . . . . . . . . . . . 141

Tecan Eight-Tip Robot (XStain BC2 Setup) . . . . . . . . . . . . . . . . . . . . . . . . 142

XStain BC2 Screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

Entering Stain Temperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Adjusting Chamber Rack to 44°C Message. . . . . . . . . . . . . . . . . . . . . . . . 144

Load BeadChips Message . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

(Infinium LIMS) Verifying Reagents and BeadChips for Coating . . . . . . . . 146

PB1 & XC4 Wash Dishes with Staining Rack . . . . . . . . . . . . . . . . . . . . . . . 148

Staining Rack Locking Arms and Tabs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

Removing Metal Clamps from Flow-Through Chamber . . . . . . . . . . . . . . 149

Washing BeadChips in PB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

Moving BeadChips from PB1 to XC4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

Staining Rack in Correct Orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

Moving BeadChip Carrier from XC4 to Tube Rack . . . . . . . . . . . . . . . . . . 152

Placing BeadChips on Tube Rack . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

Removing Staining Rack Handle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

PB1 and XC4 Wash Dishes with Staining Rack . . . . . . . . . . . . . . . . . . . . . 154

Staining Rack Locking Arms and Tabs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

Removing Metal Clamps from Flow-Through Chamber . . . . . . . . . . . . . . 155

Washing BeadChips in PB1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

Moving BeadChips from PB1 to XC4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157

Staining Rack in Correct Orientation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

Moving BeadChip Carrier from XC4 to Tube Rack . . . . . . . . . . . . . . . . . . 158

Placing BeadChips on Tube Rack . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

Moving BeadChip Carrier from XC4 to Tube Rack . . . . . . . . . . . . . . . . . . 160

Removing Staining Rack Handle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

Testing Vacuum Seal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

Part # 11280749 Rev. A

Chapter 1

Overview

10

11

8

8

11

11

4

8

2

3

Topics

Introduction

Audience and Purpose

The Infinium II Assay

The Two-Sample BeadChip

Illumina Lab Protocols

Tracking Tools

Tecan GenePaint

Illumina BeadArray Reader

Illumina BeadStudio Software

Technical Assistance

Infinium II Assay Two-Sample HC Protocols 1

2 CHAPTER 1

Overview

Introduction

The Illumina Infinium II Assay revolutionizes genotyping by streamlining sample preparation and enabling unlimited multiplexing. Using a single bead type and dual color channel approach, the Infinium II Assay scales genotyping from 10,000 to more than a million SNPs per sample, dependent only on the number of features (bead types) on the array.

The Infinium II Assay accomplishes this unlimited multiplexing by combining whole-genome amplification (WGA) sample preparation with direct, arraybased capture and enzymatic scoring of the SNP loci. Locus discrimination is provided by a combination of sequence-specific hybridization capture and array-based, single-base primer extension. A single primer is used to interrogate a SNP locus. The 3' end of the primer is positioned directly adjacent to the SNP site.

Extension of the primer incorporates a biotin nucleotide or a dinitrophenyl labeled nucleotide. (C and G nucleotides are biotin labeled; A and T nucleotides are dinitrophenyl labeled.) Signal amplification of the incorporated label further improves the overall signal-to-noise ratio of the assay.

The Illumina Infinium II Assay offers:

`

Effectively unlimited multiplexing

`

High call rate and accuracy

`

Genome-wide SNP selection

`

Single tube amplification—single chip—no PCR

`

Minimal risk of carryover contamination

`

Low DNA input mass of 750 ng

`

Walk-away automation using Tecan Genesis or Freedom EVO Robots and

Tecan GenePaint system

`

Infinium LIMS automation

`

Compatibility with both Illumina BeadLab and BeadStation systems

`

Multiple-, duo-, and single-sample BeadChip formats

`

Unlimited genotype multiplexing, scaling with the number of BeadChip features

Part # 11280749 Rev. A

Audience and Purpose 3

Audience and Purpose

This guide is for laboratory technicians running the Infinium II Assay with

Illumina two-sample Genotyping BeadChips, including the HumanHap300-

Duo, 370-Duo, HumanExon510S-Duo, and 550-Duo. These are referred to collectively as two-sample high-capacity (HC) BeadChips. The guide documents the laboratory protocols associated with the assay. Follow all of the protocols in the order shown.

Chapter 2, Two-Sample HC BeadChip Manual Protocol explains how to run

the assay manually in the lab.

Chapter 3, Two-Sample HC BeadChip Automated Protocol explains how to

automate the protocol with the aid of the Tecan eight-tip robot.

Important Note

Before following any of the procedures in this guide, read the Infinium II

Assay Lab Setup and Procedures Guide, which explains how to equip and run an Infinium II Assay laboratory. The guide includes important information on the following topics:

`

Prevention of amplification product contamination

`

Safety precautions

`

Equipment, materials, and reagents

`

Standard lab procedures

`

Robot use

`

BeadChip Imaging

`

System maintenance

`

BeadStudio controls

`

Troubleshooting

The instructions apply equally to all whole-genome genotyping chips provided by Illumina. All of the Infinium II Assay Protocol guides assume that you have already set up the laboratory space and are familiar with the standard procedures and safety precautions.

Infinium II Assay Two-Sample HC Protocols

4 CHAPTER 1

Overview

The Infinium II Assay

This section describes and illustrates the assay protocol. The assay requires only 750 ng of DNA sample as input.

Amplify DNA

The DNA samples are denatured and neutralized to prepare them for amplification.

Figure 1 Denaturing and Neutralizing DNA

See Make the AMP2 Plate on page 26 for manual processing.

See Make the AMP2 Plate on page 87 for automated processing.

Incubate DNA

The denatured DNA is isothermally amplified in an overnight step. The whole-genome amplification uniformly increases the amount of the DNA sample by several thousandfold without introducing large amounts of amplification bias.

Figure 2 Incubating DNA to Amplify

See Incubate the AMP2 Plate on page 28 for manual processing.

See Incubate the AMP2 Plate on page 99 for automated processing.

Part # 11280749 Rev. A

The Infinium II Assay 5

Fragment DNA

The amplified product is fragmented by a controlled enzymatic process that does not require gel electrophoresis. The process uses end-point fragmentation to avoid overfragmenting the sample.

Figure 3 Fragmenting DNA

See Fragment the AMP2 Plate on page 29 for manual processing.

See Fragment the AMP2 Plate on page 101 for automated process-

ing.

Precipitate DNA

After an isopropanol precipitation, the fragmented DNA is collected by centrifugation at 4ºC.

Figure 4 Precipitating DNA

See Precipitate the AMP2 Plate on page 31 for manual processing.

See Precipitate the AMP2 Plate on page 104 for automated process-

ing.

Resuspend DNA

The precipitated DNA is resuspended in hybridization buffer.

Figure 5 Resuspending DNA

See Resuspend the AMP2 Plate on page 35 for manual processing.

See Resuspend the AMP2 Plate on page 111 for automated process-

ing.

Infinium II Assay Two-Sample HC Protocols

6 CHAPTER 1

Overview

Hybridize to

BeadChip

The BeadChip is prepared for hybridization in a capillary flow-through chamber. Two samples are applied to a Beadchip divided into halves by an

IntelliHybq™ seal (or gasket). The loaded BeadChip is incubated overnight in the Illumina Hybridization Oven. The amplified and fragmented DNA samples anneal to locus-specific 50mers (covalently linked to one of over

500,000 bead types) during hybridization. One bead type corresponds to each allele per SNP locus.

(g DN A )

(id ent ical p r o b es p er b ead t y p e)

(g DN A )

Figure 6 Hybridizing DNA to BeadChip

See Hybridize HC BC2 on page 37 for manual processing.

See Hybridize HC BC2 on page 115 for automated processing.

Wash BeadChip

Unhybridized and non-specifically hybridized DNA is washed away, and the

BeadChip is prepared for staining and extension.

Figure 7 Washing BeadChip

See Wash BC2 on page 49 for manual processing.

See Wash BC2 on page 129 for automated processing.

Part # 11280749 Rev. A

The Infinium II Assay 7

Extend and Stain

(XStain)

BeadChip

Single-base extension of the oligos on the BeadChip, using the captured

DNA as a template, incorporates detectable labels on the BeadChip and determines the genotype call for the sample.

(g DN A )

A

T*

C*

G

(g DN A )

* St ain in red channel

* St ain in green channel

Figure 8 Extending and Staining BeadChip

See Single-Base Extension and Stain BC2 on page 57 for manual

processing.

See Single-Base Extension & Stain BC2 on page 138 for automated

processing.

Image BeadChip

The Illumina BeadArray Reader scans the BeadChip, using a laser to excite the fluorophore of the single-base extension product on the beads. The scanner records high-resolution images of the light emitted from the fluorophores.

Figure 9 Imaging BeadChip

See the chapter on imaging BeadChips in the Infinium II Assay Lab

Setup and Procedures Guide.

Infinium II Assay Two-Sample HC Protocols

8 CHAPTER 1

Overview

The Two-Sample BeadChip

Illumina two-sample HC BeadChips are sophisticated silicon-based array devices composed of two rows of 10–20 individual arrays on a single slide.

The two sides of the slide are separated by an IntelliHyb seal so that you can run two samples simultaneously.

Each individual array in the matrix may hold over 30,000 different oligonucleotide probe sequences. These are in turn attached to 3-micron beads assembled into the micro-wells of the BeadChip substrate. Because the micro-wells outnumber the probe sequences, multiple copies of each bead type are present in the array. This built-in redundancy improves robustness and measurement precision. The BeadChip manufacturing process includes hybridization-based quality control of each array feature, allowing consistent production of high-quality, reproducible arrays.

Illumina Lab Protocols

Illumina lab protocols are designed to promote efficiency and minimize the risk of contamination. The Infinium II Assay Lab Setup and Procedures Guide documents standard operating procedures and tools for an Infinium assay lab and explains how to set up and maintain separate pre- and post-amplification areas.

Chapter 2, Two-Sample HC BeadChip Manual Protocol and Chapter 3,

Two-Sample HC BeadChip Automated Protocol, show how to perform the

assay protocol with clearly divided pre- and post-amplification processes.

Tracking Tools

Illumina provides the following tools for sample tracking and guidance in the lab:

`

Experienced User Cards to guide you through the protocols. There are separate sets of cards for the manual and automated processes.

`

Lab Tracking Worksheet to map DNA samples to BeadChips and record the barcode of each reagent and plate used in the protocol.

`

Sample Sheet template to record information about your samples for later use in data analysis.

All of these documents are available on your Documentation CD for printing and reference.

Sample Sheet

To effectively track your samples and assay, Illumina recommends you create a Sample Sheet. The Sample Sheet will later be used by the BeadStudio application for data analysis. For instructions on data analysis, see the

BeadStudio Genotyping Module User Guide

.

Create your Sample Sheet according to the guidelines provided in Table 1.

Part # 11280749 Rev. A

Tracking Tools 9

.

Table 1 Sample Sheet Guidelines

Description

Sample_Name Name of the sample. Used only for display in the table.

Sample_Plate

The barcode of the sample plate for this sample. Used only for display in the table.

Sample_Well

The sample plate well for this sample. Used only for display in the table.

SentrixBarcode_A

The barcode of the Sentrix array product (BeadChip) to which this sample was hybridized (for Manifest A).

SentrixPosition_A

The position within the Sentrix array product to which this sample was hybridized for the manifests in your project.

Optional (O) or

Required (R)

O

O

O

R

R

Gender Male, Female, or Unknown.

Sample_Group

A group, if any, to which this sample belongs. Used for exclusion in the Final Report Wizard.

Replicate

The Sample_ID of a replicate to this sample. Used in reproducibility error calculations.

O

Parent1 The Sample_ID of this sample’s first parent.

Parent2 The Sample_ID of this sample’s second parent.

NOTES

Your sample sheet header may contain whatever information you choose.

Your sample sheet may contain any number of columns you choose.

Your sample sheet must be in a comma-delimited (.csv) file format.

Save the sample sheet under any name you wish; for example, the user-defined experiment name.

O

O

O

O

Figure 10 provides an example of the Sample Sheet format. Your

Documentation CD includes an electronic, read-only Sample Sheet template file (Sample Sheet Template.csv) that you can copy and use.

Infinium II Assay Two-Sample HC Protocols

10 CHAPTER 1

Overview

Figure 10 Sample Sheet Example

Tecan GenePaint

The Infinium II Assay uses Tecan's GenePaint automated slide processor to process BeadChips. The GenePaint system employs a capillary gap flowthrough chamber to enable reagent entrapment and exchange over the

BeadChip’s active surface. Washing, blocking, extension, and signal amplification are all performed by simple reagent additions to the flow cell.

Addition of a new reagent displaces the entrapped reagent from the flow cell. For maximum flexibility, these additions can be performed either manually or via the Tecan Genesis robot. The optional automated robotic processing and single-use reagent tube barcoding assure maximum consistency from slide to slide.

Part # 11280749 Rev. A

Illumina BeadArray Reader 11

Illumina BeadArray Reader

BeadChips are imaged using the Illumina BeadArray Reader, a two-channel,

0.8 μm resolution confocal laser scanner. The BeadArray Reader scans

BeadChips at two wavelengths simultaneously and creates an image file for each channel (i.e., two per array).

The BeadScan software determines intensity values for each bead type and creates data files for each color. BeadStudio uses this data file in conjunction with the individual bead pool map (*.bpm) file to analyze the data from the assay.

For instructions on imaging the BeadChip, see the Infinium II Assay Lab

Setup and Procedures Guide. For instructions on using the BeadArray

Reader, see the

Illumina BeadArray Reader User Guide

.

Illumina BeadStudio Software

The BeadStudio software package included with the Illumina Infinium II Assay system extracts whole-genome genotyping data from data files created by the Illumina BeadArray Reader. You can export the resulting files to most standard databases and gene expression analysis programs.

For instructions on data analysis, see the

BeadStudio Genotyping Module

User Guide

.

Technical Assistance

For technical assistance, contact Illumina Customer Support.

Table 2 Illumina Customer Support Contacts

Contact

Toll-free Customer Hotline

International Customer Hotline

Illumina Website

Number

1-800-809-ILMN (1-800-809-4566)

1-858-202-ILMN (1-858-202-4566) www.illumina.com

Infinium II Assay Two-Sample HC Protocols

12 CHAPTER 1

Overview

Part # 11280749 Rev. A

Chapter 2

Two-Sample HC BeadChip

Manual Protocol

37

49

57

71

28

29

31

35

19

22

25

26

14

14

16

17

Topics

Introduction

Two-Sample BeadChip Manual Workflow

Equipment, Materials, and Reagents

Make Standard DNA Plate (Optional/Infinium LIMS)

Make Quant (Optional/LIMS)

Read Quant (Optional/LIMS)

Prepare the WG#-DNA Plate (Optional)

Make the AMP2 Plate

Incubate the AMP2 Plate

Fragment the AMP2 Plate

Precipitate the AMP2 Plate

Resuspend the AMP2 Plate

Hybridize HC BC2

Wash BC2

Single-Base Extension and Stain BC2

Image BC2

Infinium II Assay Two-Sample HC Protocols 13

14 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Introduction

This chapter describes pre- and post-amplification manual laboratory protocols for Illumina two-sample Genotyping BeadChips. Follow the protocols in the order shown.

Two-Sample BeadChip Manual Workflow

Figure 11 graphically represents the Illumina Infinium II Assay manual

workflow for the two-sample Genotyping BeadChip. These protocols describe the procedure for preparing eight DNA samples. To process 48 or

96 samples, scale up the protocols accordingly.

Part # 11280749 Rev. A

Day 1

Make AMP2

Hands-on:

~20 min/8 samples

Reagents

0.1N NaOH

MP1

AMM

Output

AMP2 Plate

Incubate AMP2

Incubation: 20–24 hours

Output

AMP2 Plate with

Amplified DNA

Day 2

Fragment AMP2

Hands-on: ~30 min

Incubation: 60 min

Reagents

FRG

Output

AMP2 Plate

Precip AMP2

Hands-on: ~30 min/plate

Incubation: 50 min

Dry: 60 min

Reagents

2-propanol

PA1

Output

AMP2 Plate

Resuspend AMP2

Hands-on: ~30 min

Incubation: 60 min

Reagents

RA1

Output

AMP2 Plate

Two-Sample BeadChip Manual Workflow 15

Day 3

Wash BC2

Hands-on:

~20 min/4 BeadChips

Reagents

WB1

PB1

Output

BeadChip

XStain BC2

Hands-on: ~3 hours

Dry: 60 min

Reagents

RA1

95% Formamide /

1 mM EDTA

PB1

XC1

XC2

XC3

XC4

TEM

LTM

ATM

Output

BeadChip

Image BC2

Scan: 45 min/BeadChip

Output

Image and Data Files

Hyb HC BC2

Hands-on:

~30 min/8 samples

Incubation: 16–24 hours

Reagents

PB2

Output

BeadChip

Figure 11 Two-Sample BeadChip Manual Workflow

Infinium II Assay Two-Sample HC Protocols

Pre-Amp

Post-Amp

Optional stop, cold storage

Overnight incubation

16 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Equipment, Materials, and Reagents

These items are specific to the two-sample, manual BeadChip assay. For a list of other equipment, materials, and reagents needed in an Infinium II Assay lab, see the Infinium II Assay Lab Setup and Procedures Guide.

Equipment Illumina-Supplied

Table 3 Illumina-Supplied Equipment

Item

Multi-Sample BeadChip Alignment Fixture

Catalog #

Illumina part # 218528

Materials User-Supplied

Table 4 User-Supplied Equipment

Item

Vacuum desiccator (1 per 8 BeadChips processed simultaneously)

Vacuum tubing

Tube racks for vacuum desiccators (must fit internal dimensions of vacuum desiccator)

Vacuum source (greater than 508 mm Hg (0.68 bar))

Vacuum gauge for vacuum desiccator (Recommended)

Source

VWR Catalog # 24988-197

VWR Catalog # 62995-335

VWR Catalog # 60916-748

Illumina-Supplied

`

AMP2 barcode labels

`

WG#-DNA barcode labels

Reagents Illumina-Supplied

Table 5 Illumina-Supplied Reagents

Item

MP1—Neutralization solution

AMM—Amplification Master Mix

FRG—Fragmentation solution

PA1—Precipitation solution

Part #

11190751

11192044

11190022

11190031

Part # 11280749 Rev. A

Make Standard DNA Plate (Optional/Infinium LIMS) 17

Table 5 Illumina-Supplied Reagents

Item

RA1—Resuspension, hybridization, and wash solution

PB1—Reagent used to prepare BeadChips for hybridization

PB2—Humidifying buffer used during hybridization

XC1—XStain BeadChip solution 1

XC2—XStain BeadChip solution 2

TEM—Two-Color Extension Master Mix

XC3 (80 ml)—XStain BeadChip solution 3

XC3 (240 ml)—XStain BeadChip solution 3

LTM—Labeling Two-Color Master Mix

ATM—Anti-Stain Two-Color Master Mix

XC4—XStain BeadChip solution 4

Part #

11191914

11191922

11191130

11208288

11208296

11208309

11208392

11208421

11208325

11208317

11208430

Make Standard DNA Plate (Optional/Infinium LIMS)

This process creates a Standard DNA plate with specific concentrations of

DNA in the wells. Use this plate as input into the Make Quant process. If your

DNA has already been quantified and you are not running Infinium LIMS

(Laboratory Information Management System), you do not have to perform this step.

For information about how to use Infinium LIMS, see the

Infinium II LIMS User

Guide

.

Consumables

Item Quantity Storage

Lambda DNA

1X TE

See formula below

(10 mM Tris-HCl pH8.0, 1 mM EDTA)

Room temperature

96-well microtiter plate (MIDI) 1 per 96 samples

Supplied By

User

User

User

Preparation

`

Remove PicoGreen reagent from freezer and thaw at room temperature for 60 minutes in a light-impermeable container.

`

Label a 96-well 0.65 ml MIDI plate “Standard QNT.”

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18 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Steps Make Standard QNT MIDI Plate

In this process, you create a Standard QNT plate with serial dilutions of stock

Lambda DNA in the wells of column 1.

1. Place stock Lambda DNA in well A1 of the Standard QNT MIDI plate and dilute it to 75 ng/μl in a final volume of 233.3 μl.

a. Use the following formula to calculate the amount of stock Lambda

DNA to add to A1:

(233.3

μl

) X (75 ng/μl)

(stock Lambda DNA concentration)

= μl

of stock

Lambda

DNA to add to A1 b. Dilute the stock DNA in well A1 using the following formula:

μl of 1X TE to add to A1 =

233.3

μ well A1 l -

μl

of stock

Lambda

DNA in

2. Add 66.7 μl 1X TE to well B of column 1 of the same plate.

3. Add 100 μl 1X TE to wells C, D, E, F, G, and H of column 1 of the same plate.

λ

DNA 1X TE

75 ng/µL

66.7

µL

100

µL

100

µL

100

µL

100

µL

100

µL

100

µL

A B C D E F G H

Figure 12 MIDI Plate Wells

MIDI Plate

4. Pipette the contents of A1 up and down 10 times to mix.

5. Transfer 133.3 μl of Lambda DNA from well A1 into well B1, and then pipette the contents of well B1 up and down 10 times.

6. Change pipette tips. Transfer 100 μl from well B1 into well C1, and then pipette the contents of well C1 up and down 10 times.

7. Change pipette tips. Transfer 100 μl from well C1 into well D1, and then pipette the contents of well D1 up and down 10 times.

8. Change pipette tips. Transfer 100 μl from well D1 into well E1, and then pipette the contents of well E1 up and down 10 times.

9. Change pipette tips. Transfer 100 μl from well E1 into well F1, and then pipette mix the contents of well F1 up and down 10 times.

Part # 11280749 Rev. A

Make Quant (Optional/LIMS) 19

10. Change pipette tips. Transfer 100 μl from well F1 into well G1, and then pipette the contents of well G1 up and down 10 times.

11. Do not transfer solution from well G1 to well H1. Well H1 serves as the blank 0 ng/μl Lambda DNA.

Table 6 Concentration of Lambda DNA Standards

Row-Column

A1

B1

C1

D1

E1

F1

G1

H1

Conc. (ng/μl)

75

50

25

12.5

6.25

3.125

1.5625

0

Final Volume in well (μl)

100

100

100

100

100

100

200

100

12. Cover the plate with a cap mat.

13. Do one of the following:

• Store the plate at 4°C for future use.

•

Proceed to Make Quant (Optional/LIMS).

Make Quant (Optional/LIMS)

In this process, you create one to three QNT plates for use in the Molecular

Dynamics Fluorometer (if available). The fluorometer quantifies the DNA present in the sample and enters that data into Infinium LIMS. Quantification ensures that there is enough sample DNA to generate good data. Use this procedure if you are running Infinium LIMS or if you have a fluorometer in your lab.

Consumables User-Supplied

Item

PicoGreen dsDNA quantitation reagent, thawed

1X TE (10 mM Tris-HCl pH

8.0, 1 mM EDTA)

Quantity Storage Supplied By

User

Room temperature

User

Infinium II Assay Two-Sample HC Protocols

20 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Item Quantity

DNA samples, normalized to

50 ng/μl

8, 16, 24, 32, 48, or

96 in up to 3

WG#-DNA plates

96-well microtiter plate (MIDI) 1 per 96 samples

Fluotrac 200 (96-well black flat-bottom) plate

2 per 96 samples

Storage Supplied By

User

User

User

NOTE

PicoGreen is susceptible to differential contaminants. False positives may occur for whole-genome amplification.

Therefore, it is important to quantify the input into the whole-genome amplification reaction.

Preparation

`

For each WG#-DNA plate you plan to quantify, place a QNT barcode label on a new black Fluotrac plate. Position the label on the skirt of the plate, where the manufacturer’s name appears.

Prepare PicoGreen Dilution

CAUTION

PicoGreen reagent degrades quickly in the presence of light. Do not use glass containers for PicoGreen reagent.

1. Wrap aluminum foil around a sterile plastic container to prevent light penetration.

2. Make a 1:200 dilution of PicoGreen to 1X TE in the sterile plastic container.

Table 7 PicoGreen Reagent Volumes

# QNT Plates

1

2

3

PicoGreen Volume (μl)

125

230

325

1X TE Volume (ml)

25

45

65

You can prepare dilutions for up to three sample plates at a time.

3. Mix dilution thoroughly.

4. Pour the PicoGreen dilution into a sterile multichannel-pipette reservoir.

Part # 11280749 Rev. A

Make Quant (Optional/LIMS) 21

5. Transfer 195 μl PicoGreen dilution to each well in columns 1 and 2 of a

new 96-well black flat-bottom plate (Figure 13). This is the Standard QNT

plate.

Standard QNT Plate with PicoGreen

= 195 µl Picogreen/1X TE Dilution

+ 2 µl Lambda DNA Serial Dilutions

Figure 13 Standard QNT Plate

6. Add 2 μl of stock Lambda DNA from the Standard DNA plate to each well in columns 1 and 2 of the Standard QNT plate. Transfer from well A1 in the Standard DNA plate to well A1 in the Standard QNT plate, and so on for the rest of the wells.

7. Pipet the contents of the Standard QNT plate up and down several times.

8. Cover the plate with an aluminum adhesive seal and label as “Standard

QNT Plate.”

9. Transfer 195 μl PicoGreen dilution to all 96 wells of the QNT plate (Figure

14).

Sample QNT Plate with PicoGreen

= 195 µl Picogreen/1X TE Dilution

+ 2 µl DNA Sample

Figure 14 QNT Plate

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22 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

10. Add 2 μl of sample DNA from the WG#-DNA plate to each well of the black, flat-bottom QNT plate. Transfer from well A1 in the WG#-DNA plate to well A1 in the QNT plate, and so on for the rest of the wells.

11. Pipette the contents of the QNT plate up and down several times.

12. Cover the QNT plate with an aluminum adhesive seal.

13. On the lab tracking worksheet, record:

• Date/Time

• Operator

• The QNT barcode that corresponds to each WG#-DNA barcode

• The Standard QNT plate that corresponds to each Standard DNA plate.

14. Proceed to Read Quant (Optional/LIMS) on page 22.

Read Quant (Optional/LIMS)

In this process, you use a fluorometer along with the Infinium Fluorometry

Analysis software to interpret the quantified DNA in the QNT plate(s) and obtain the exact concentration of DNA in the sample. This information is saved with the other project data in Infinium LIMS. Illumina recommends using a fluorometer because fluorometry provides DNA-specific quantification. Spectrophotometry may also measure RNA, yielding values that are too high.

For information about how to use Infinium LIMS, see the Infinium II LIMS User

Guide.

Read Quant

1. Turn on the fluorometer.

2. Open the Infinium Fluorometry Analysis software.

Part # 11280749 Rev. A

Read Quant (Optional/LIMS) 23

Figure 15 Infinium Fluorometry Analysis Opening Screen

3. Select Reader Tasks | Read Quant.

4. Select the Use Barcodes check box.

5. Click Read.

6. When prompted, log in to the Infinium LIMS database.

7. When asked if you want to read a new Standard QNT plate, click Yes.

Load the Standard QNT plate in the open drawer of the fluorometer.

8. When the fluorometer finishes reading the Standard QNT data, unload the plate.

9. When prompted, enter the number of QNT plates you want to read. You can read up to three plates. Click OK.

Figure 16 Number of QNT Plates Dialog Box

10. When prompted, enter the QNT plate barcode. Load the QNT plate in the open drawer of the fluorometer and click OK.

Figure 17 Plate Barcode Dialog Box

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24 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

The Fluorometry Analysis screen fills in with information about the fluorescence in the wells.

Figure 18 Fluorescence Data

Microsoft Excel opens automatically at the same time and displays the quant data for the QNT plate. There are three tabs in the file:

• SQNT_STD—Plots the RF values against the concentration (ng/μl).

• QNT—Plots the concentration (ng/μl) for each well.

• Data—Compares the data from the Standard QNT plate to the QNT plate you just read.

The Infinium Fluorometer Analysis software prompts you to indicate whether you wish to use the QNT data shown in the Excel file.

11. Do one of the following:

• Click Yes to send the data to Infinium LIMS. In Infinium LIMS, the

QNT plate moves into the Make AMP2 queue.

• Click No to delete the quant data. You can read the quant data again for the same plate.

12. If you entered more than one QNT plate to read, repeat steps 10 and 11 for each additional plate.

13. Discard the QNT plates and reagents in accordance with facility requirements.

14. Proceed to Make the AMP2 Plate on page 26.

Part # 11280749 Rev. A

Prepare the WG#-DNA Plate (Optional) 25

Prepare the WG#-DNA Plate (Optional)

DNA samples must be 50 ng/μl, resuspended in TE (10 mM Tris, 1 mM

EDTA).

1. Retrieve your DNA samples and thaw to room temperature (22°C).

2. Apply a WG#-DNA barcode label to a new 0.8 ml microtiter storage plate (MIDI) or a new 0.2 ml skirted microplate (TCY).

3. Dispense DNA according to Figure 19:

• For MIDI plate: 40 μl DNA sample to each well

• For TCY plate: 30 μl DNA sample to each well

S a m p le s

1

t o

8

S a m p le s

t

9 o

1

6

S a m p le s

1

7

t

2 o

4

WG#-DNA Plate

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Figure 19 Distributing Sample to WG#-DNA Plate

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26 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Make the AMP2 Plate

This process creates a AMP2 plate for DNA amplification. The DNA sample is denatured with NaOH and then neutralized with MP1 reagent. The last reagent added is AMM (Amplification Master Mix).

Figure 20 Denaturing and Neutralizing DNA

Estimated Time

Hands-on time: ~20 minutes per 8 samples

Incubation time: ~20–24 hours

Consumables

Item

MP1

AMM

0.1N NaOH

Quantity Storage

1 tube per 8 samples -20°C

1 tube per 8 samples -20°C

15

μl

for 8–24 samples

4°C

96-well 0.8 ml microtiter plate

(MIDI)

1 plate per

24 samples

WG#-DNA plate with 96 DNA samples (50 ng/μl)

1 plate

Supplied By

Illumina

Illumina

User

User

User

Preparation

`

Preheat the Illumina Hybridization Oven in the post-amp area to 37°C and allow the temperature to equilibrate.

`

Apply an AMP2 barcode label to a new MIDI plate.

`

Thaw MP1 and AMM tubes to room temperature (22°C). Gently invert to mix, then pulse centrifuge to 280 xg.

`

Thaw DNA samples to room temperature (22°C).

`

Enter the Sample_Name (optional) and Sample_Plate for each

Sample_Well in the sample sheet.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• WG#-DNA plate barcode

Part # 11280749 Rev. A

Make the AMP2 Plate 27

• AMP2 plate barcode

• MP1 tube barcode(s)

• AMM tube barcode(s)

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Steps

1. If you do not already have a WG#-DNA plate, dispense DNA into either a:

• MIDI plate: 40 μl to each WG#-DNA plate well

• TCY plate: 30 μl to each WG#-DNA plate well

Apply a barcode label to the new WG#-DNA plate.

2. Vortex the WG#-DNA plate at 1600 rpm (actual vortex speed) for

1 minute.

3. Centrifuge to 280 xg for 1 minute.

4. Transfer 15 μl DNA sample, normalized to 50 ng/μl, into each well in the following AMP2 plate columns:

• Column 1 (8 samples)

• Columns 1 and 5 (16 samples)

• Columns 1, 5, and 9 (24 samples)

5. Dispense 15 μl 0.1N NaOH into each well that contains DNA (Figure 21).

S a m p le s

1

t

8 o

S a m p le s

9

t

1 o

6

S a m p le s

1

7

t o

2

4

WG#-DNA Plate

C

D

A

B

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Figure 21 Distributing Sample to AMP2 Plate Wells

E

F

G

H

A

B

C

D

S a m p le s

t

1 o

8

S a m p le s

9

t o

1

6

S a m p le s

1

7

t

2 o

4

AMP2 Plate

1 2 3 4 5 6 7 8 9 10 11 12

CAUTION

To ensure optimal performance of sample and lab equipment, use aerosol filter tips when pipetting DNA.

6. Incubate for 10 minutes at room temperature (22°C).

Infinium II Assay Two-Sample HC Protocols

28 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

7. Record each DNA sample in the lab tracking worksheet.

8. Dispense 270 μl MP1 into each well containing sample, taking care to avoid cross-contamination.

9. Dispense 300 μl AMM into each well containing sample, taking care to avoid cross-contamination.

10. Seal the plate with a cap mat.

11. Invert the sealed plate at least 10 times to mix contents.

12. Pulse centrifuge to 280 xg.

13. Discard unused reagents in accordance with facility standards.

14. Proceed immediately to Incubate the AMP2 Plate on page 28.

Incubate the AMP2 Plate

This process uniformly amplifies the genomic DNA, generating a sufficient quantity of each individual DNA sample to be used twice in the Two-Sample

Infinium II Assay.

Figure 22 Incubating DNA to Amplify

Incubation Time

~20–24 hours.

Steps

1. Incubate the AMP2 plate in the Illumina Hybridization Oven for at least

20 and no more than 24 hours at 37°C.

2. On the lab tracking worksheet, record the start and stop times.

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

3. Proceed to Fragment the AMP2 Plate on page 29.

Part # 11280749 Rev. A

Fragment the AMP2 Plate 29

Fragment the AMP2 Plate

This process enzymatically fragments the amplified DNA samples. An endpoint fragmentation is used to prevent over-fragmentation.

Figure 23 Fragmenting DNA

Estimated Time

Hands-on time: ~30 minutes

Incubation time: 1 hour

Consumables

Item

FRG

Quantity Storage

1 tube per 8 samples -20°C

Supplied By

Illumina

Preparation

`

Preheat the heat block with the MIDI plate insert to 37°C.

`

Thaw the FRG tube to room temperature (22°C). Invert several times to mix contents. Pulse centrifuge to 280 xg for 1 minute.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• FRG tube barcode(s)

`

Remove the AMP2 plate from the Illumina Hybridization Oven.

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Steps

1. Centrifuge the plate to 50 xg for 1 minute.

2. Remove the cap mat.

NOTE

When you remove a cap mat, set it aside, upside down, in a safe location for use later in the protocol. When you place the cap mat back on the plate, be sure to match it to its original plate and orient it correctly.

Infinium II Assay Two-Sample HC Protocols

30 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

3. Thoroughly pipette-mix all wells containing sample to evenly distribute precipitate.

4. Split the sample into 3 additional wells, for a total of 4 wells per sample.

Each well should contain 150 μl.

For example, move 150 μl sample from A1 into A2, A3, and A4.

• Divide DNA sample in A1 into A2, A3, and A4.

• Divide DNA sample in A5 into A6, A7, and A8.

• Divide DNA sample in A9 into A10, A11, and A12.

Follow this pattern for rows B–H, columns 1, 5, and 9 (see Figure 24).

S a m p le s

t

1 o

8

S a m p le s

t

9 o

1

6

S a m p le s

1

7

t o

2

4

AMP2 Plate

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Figure 24 Distributing Sample in AMP2 Plate

5. Dispense 50 μl FRG to each well containing sample.

6. Seal the AMP2 plate with the cap mat.

7. Place the sealed plate on the vortexer and secure it with the provided

Velcro straps (Figure 25).

Figure 25 Securing Plates to Vortexer Platform with Velcro Straps

Part # 11280749 Rev. A

Precipitate the AMP2 Plate 31

8. Vortex the AMP2 plate at 1600 rpm for 1 minute.

9. Centrifuge the plate to 50 xg for 1 minute at 22°C.

10. Incubate the sealed plate on the 37°C heat block for 1 hour.

11. On the lab tracking worksheet, record the start and stop times.

12. Discard unused reagents in accordance with facility standards.

13. Do one of the following:

•

Proceed to Precipitate the AMP2 Plate on page 31. Leave the plate

in 37°C heat block until setup is complete.

• Store the sealed AMP2 plate at -20°C if you do not plan to proceed to the next step immediately.

This is a good stopping point in the process.

Precipitate the AMP2 Plate

Add PA1 and 2-propanol to the AMP2 plate to precipitate the DNA samples.

Figure 26 Precipitating DNA

Estimated Time

Hands-on time: ~30 minutes per plate

Incubation time: 2 hours

Consumables

Item

PA1

100% 2-propanol

Quantity Storage

1 tube per 8 samples 4°C

12 ml per 8 samples

40 ml per 24 samples

Room temperature

Supplied By

Illumina

User

Preparation

`

Do one of the following:

• If you froze the AMP2 plate after fragmentation, thaw it to room temperature (22°C). Centrifuge to 280 xg for 1 minute.

Infinium II Assay Two-Sample HC Protocols

32 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

• If you continued immediately from Fragment AMP2, leave the plate in the 37°C heat block until setup is complete.

`

Thaw PA1 to room temperature (22°C). Centrifuge to 280 xg for

1 minute.

`

Preheat the heat block to 37°C, if it is not already.

`

Turn on the heat sealer.

`

In preparation for the 4°C spin, set the centrifuge to 4°C.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• PA1 tube barcode(s)

• 2-propanol lot number and date opened

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Steps

1. Dispense 100 μl PA1 to each well containing sample.

2. Seal the plate with the cap mat.

3. Vortex the plate at 1600 rpm for 1 minute.

4. Centrifuge to 50 xg at 22°C for 1 minute.

5. Incubate at 37°C for 5 minutes.

6. Add 300 μl 100% 2-propanol to each well containing sample.

7. Carefully heat-seal the storage plate, taking care not to shake the plate in any way until the heat-seal is fully seated.

8. Invert at least 10 times to mix contents.

9. Incubate at 4°C for 30 minutes.

10. Place the sealed AMP2 plate in the centrifuge opposite another plate of

equal weight (Figure 27).

Part # 11280749 Rev. A

Precipitate the AMP2 Plate 33

Plate of Equal Weight for Balance

Sealed AMP2 Plate

Figure 27 Balancing AMP2 Plate in Centrifuge

11. Centrifuge to 3000 xg at 4°C for 20 minutes. When the spin finishes, immediately remove the AMP2 plate from the centrifuge.

Perform the next step immediately, to avoid dislodging the blue pellet. If any delay occurs, repeat the 20-minute centrifugation before proceeding.

12. Remove the heat-seal and discard it.

13. Decant the supernatant by quickly inverting the AMP2 plate and smacking it down onto an absorbent pad appropriate for 2-propanol disposal.

14. Tap the plate firmly on the pad several times over a period of 1 minute or until all wells are completely devoid of liquid.

CAUTION

Keep plate inverted. To ensure optimal performance while decanting, do not allow supernatant in wells to pour into other wells.

15. Place the inverted, uncovered plate on a tube rack for 1 hour at room

temperature to air dry the pellet (Figure 28).

At this point, blue pellets should be present at the bottoms of the wells.

16. On the lab tracking worksheet, record the start and stop times.

Infinium II Assay Two-Sample HC Protocols

34 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Figure 28 Uncovered AMP2 Plate Inverted for Air Drying

CAUTION

Do not over-dry the pellet. Pellets that are over-dried will be difficult to resuspend. Poorly resuspended samples will lead to poor genotyping results.

17. Discard unused reagents in accordance with facility standards.

18. Do one of the following:

•

Proceed to Resuspend the AMP2 Plate on page 35.

• Heat-seal the AMP2 plate and store it at -20°C for up to 24 hours or

-80°C for long-term storage.

This is a good stopping point in the process.

Part # 11280749 Rev. A

Resuspend the AMP2 Plate 35

Resuspend the AMP2 Plate

Add RA1 to the AMP2 plate to resuspend the precipitated DNA samples.

Figure 29 Resuspending DNA

Estimated Time

Hands-on time: ~30 minutes

Incubation time: 1 hour

Consumables

Item

RA1

Quantity

Bottle (1.5 ml)

Storage

-20°C

Supplied By

Illumina

This protocol involves the use of an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. Refer to the Infinium Assay

MSDS on your Documentation CD for complete information.

Preparation

`

RA1 is shipped frozen. Gradually warm the RA1 reagent to room temperature, preferably in a 20–25°C water bath. Gently mix to dissolve any crystals that may be present.

`

If you stored the AMP2 plate at -20°C, thaw it to room temperature.

Remove the cap mat and discard it.

`

Preheat the Illumina Hybridization Oven to 48°C.

`

Turn on the heat sealer to preheat. Allow 20 minutes.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• RA1 bottle barcode(s)

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Infinium II Assay Two-Sample HC Protocols

36 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Steps

1. Add 42 μl RA1 to each well of the AMP2 plate containing a DNA pellet.

Reserve leftover reagent for Hyb HC BC2 and XStain BC2.

2. Heat-seal the AMP2 plate with a foil seal.

3. Incubate it in the Illumina Hybridization Oven for 1 hour at 48°C.

4. On the lab tracking worksheet, record the start and stop times.

5. Vortex the plate at 1800 rpm for 1 minute.

6. Pulse centrifuge to 280 xg.

If you stored the pellets at -20°C for more than 72 hours after the Precip

AMP2 process, you may need to repeat steps 3 to 6 until the pellets are completely resuspended.

7. Discard unused reagents in accordance with facility standards.

8. Do one of the following:

•

Proceed to Hybridize HC BC2 on page 37. If you plan to do so

immediately, it is safe to leave the RA1 at room temperature.

• Store the sealed AMP2 plate and the RA1 at -20°C (-80°C if storing for more than 24 hours).

This is a good stopping point in the process.

Part # 11280749 Rev. A

Hybridize HC BC2 37

Hybridize HC BC2

In this process, you dispense the resuspended, denatured DNA samples onto

BeadChips. First, the BeadChips are placed into Hyb Chamber inserts. Each slide is loaded with two DNA samples. Place the inserts into the Hyb

Chambers. Incubate the Hyb Chambers in the Illumina Hybridization Oven for 16–24 hours at 48°C.

(g DN A )

(id ent ical p r o b es p er b ead t y p e)

(g DN A )

Figure 30 Hybridizing DNA to BeadChip

Estimated Time

Hands-on time:

• ~30 minutes for 8 samples

• ~1 hour for 24 samples

Incubation time: 16–24 hours

Consumables

Item

PB2

Quantity

(per 8 Samples)

1 tube

BeadChips

Hyb Chambers

Hyb Chamber gaskets

Hyb Chamber inserts

4

1 per 8 samples

(4 BeadChips)

1 per 8 samples

(4 BeadChips)

4

Storage Supplied By

Room temperature

Illumina

Illumina

Illumina

Illumina

Illumina

NOTE

Thaw all reagents completely at room temperature (22°C) and allow to equilibrate. Once thawed, gently invert each tube several times to thoroughly mix the reagent. Pulse centrifuge each tube to 280 xg to eliminate bubbles and collect reagent at the bottom of the tube.

Preparation

`

Preheat the heat block to 95°C.

`

Preheat the Illumina Hybridization Oven to 48°C and set the rocker speed to 5.

Infinium II Assay Two-Sample HC Protocols

38 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

`

Prepare the Illumina Hybridization Oven as follows: a. Preheat the oven to 48°C:

—

Press the "F" button once to change the display to TSET.

— Press the "S" button to enter the set-temperature mode, and then use the Increment/Decrement dial to set the oven to 48°C.

— Press the "S" button again to set 48°C as the temperature.

b. Set the rocker speed to 5:

—

Press the "F" button twice until SPd is indicated on the display.

— Press the "S" button to enter the rocker speed mode.

— Use the Increment/Decrement dial to set the rocker speed to "5".

— Press the "S" button again.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• PB2 tube barcode(s)

`

Calibrate the Illumina Hybridization Oven with the Full-Scale Plus digital thermometer supplied with your system.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• PB2 tube barcode(s)

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Steps

This protocol includes the following steps:

`

Assemble Hyb Chambers on page 38

`

Hyb HC BeadChip on page 41

`

Load BeadChip on page 42

`

Set Up HC BC2 for Hyb on page 44

NOTE

Perform the Hyb Chamber assembly near the Hyb Oven to minimize the distance you need to move the BeadChiploaded Hyb Chamber. Take care to keep the Hyb Chamber steady and level when lifting and moving. Avoid shaking and keep the Hyb Chamber parallel to the lab bench at all times.

Assemble Hyb Chambers

1. For every 4 BeadChips, place the following items on the bench top

(Figure 31):

• BeadChip Hyb Chambers (1)

Part # 11280749 Rev. A

Hybridize HC BC2 39

• Hyb Chamber Gaskets (1)

• BeadChip Hyb Chamber Inserts (4)

Hyb Chamber

Gasket

Hyb Chamber

Hyb Chamber

Inserts

Figure 31 BeadChip Hyb Cartridge Components

Barcode Ridges

2. Place the BeadChip Hyb Chamber gasket into the BeadChip Hyb

Chambers: a. Match the wider edge of the Hyb Chamber gasket to the barcoderidge side of the Hyb Chamber (Figure 32).

Wider Edge

Reservoirs Narrower Edge

Figure 32 Hyb Chamber and Gasket

b. Lay the gasket into the Hyb Chamber (Figure 33), and then press it down all around.

Figure 33 Placing Gasket into Hyb Chamber

Infinium II Assay Two-Sample HC Protocols

40 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol c.

Make sure the Hyb Chamber gasket is properly seated (Figure 34).

Figure 34 Hyb Chamber with Gasket in Place

3. Dispense 200 μl PB2 into each of the humidifying buffer reservoirs in the

Hyb Chamber (Figure 35).

Figure 35 Dispensing PB2 into Hyb Chamber Reservoirs

4. Close and lock the BeadChip Hyb Chamber lid (Figure 36).

a. Seat the lid securely on the bottom plate.

b. Snap two clamps shut, diagonally across from each other.

c.

Snap the other two clamps.

Part # 11280749 Rev. A

Hybridize HC BC2 41

Figure 36 Sealing the Hyb Chamber

5. Leave the closed Hyb Chamber on the bench at room temperature until the BeadChips are loaded with DNA sample.

Hyb HC BeadChip

In this step, you will denature and consolidate the DNA sample.

1. Place the resuspended AMP2 plate on the heat block to denature the samples at 95°C for 20 minutes.

CAUTION

Do not unpackage the BeadChips until you are ready to begin hybridization.

2. Remove the BeadChips from 4°C storage but do not unpackage.

3. After the 20-minute incubation, pulse centrifuge the AMP2 plate to

280 xg. Remove the foil seal.

4. Combine the four separate wells back into the original well. Refer to

Figure 37:

a. Combine contents of wells A2, A3, and A4 back into well A1.

b. Combine contents of wells A6, A7, and A8 back into well A5.

c.

Combine contents of wells A10, A11, and A12 back into well A9.

d. Repeat for rows B–H.

Infinium II Assay Two-Sample HC Protocols

42 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

S a m p le s

t

1 o

8

S a m p le s

t

9 o

1

6

S a m p le s

1

7

t o

2

4

AMP2 Plate

C

D

E

F

A

B

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Figure 37 Consolidating Sample Back Into Original Sample Well

Load BeadChip

1. Just before loading DNA samples, remove all BeadChips from their packages.

CAUTION

Hold the BeadChip by the ends with your thumb and forefinger (thumb at the barcode end). Do not hold the

BeadChip by the sides near the sample inlets. Avoid contacting the beadstripe area and sample inlets.

2. Place each BeadChip in a Hyb Chamber insert, orienting the barcode end so that it matches the barcode symbol on the Hyb Chamber insert

(Figure 38). Make sure you place each BeadChip into the Hyb Chamber

insert prior to loading the DNA sample.

Figure 38 Placing BeadChips into Hyb Chamber Inserts

3. Dispense 84 μl of each DNA sample into the appropriate BeadChip inlet

port according to the lab tracking worksheet. See Figure 39 for a

diagram and Figure 40 for a photo of the BeadChip.

Part # 11280749 Rev. A

Hybridize HC BC2 43

CAUTION

Load samples by directly placing pipette tips to the array surface. To avoid evaporation, proceed immediately to the next step as soon as all arrays have received sample.

a. Load samples in the A1 and B1 wells of the AMP2 plate into the first

BeadChip.

b. Load samples in C1 and D1 into the second BeadChip. c.

Load samples in E1 and F1 into the third BeadChip. d. Load samples in G1 and H1 into the fourth BeadChip.

Repeat the same pattern to transfer sample from column 5 to BeadChips

5–8, and from column 9 to BeadChips 9–12.

2 MP 56789-A 11234 WG

6

5

8

7

4

3

2

1

12

11

10

9

A B C D E F G H

A1 B1 C1 D1 E1 F1 G1 H1

Two-Sample

BeadChips

1, 5, 9

Two-Sample

BeadChips

2, 6, 10

Two-Sample

BeadChips

3, 7, 11

Figure 39 Distributing Sample to BeadChips (Pattern)

Two-Sample

BeadChips

4, 8, 12

Infinium II Assay Two-Sample HC Protocols

44 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Figure 40 Dispensing Sample onto BeadChip (Photo)

4. On the lab tracking worksheet, record the BeadChip barcodes associated with each sample well.

5. Visually inspect all sections of the BeadChips to ensure the DNA sample covers all of each bead stripe. Record any sections that are not completely covered.

Set Up HC BC2 for Hyb

CAUTION

For optimal performance, take care to keep the Hyb

Chamber inserts containing BeadChips steady and level when lifting or moving. Avoid shaking and keep parallel to the lab bench at all times. Do not hold by the sides near the sample inlets.

1. Load the Hyb Chamber inserts containing BeadChips into the Illumina

Hyb Chamber (Figure 41). Position the barcode end over the ridges indicated on the Hyb Chamber.

Figure 41 Placing Hyb Chamber Inserts into Hyb Chamber

2. Place the back side of the lid onto the Hyb Chamber and then slowly bring down the front end to avoid dislodging the Hyb Chamber inserts

(Figure 42).

Part # 11280749 Rev. A

3. Close the clamps on both sides of the Hyb Chamber.

Hybridize HC BC2 45

Figure 42 Securing Hyb Chamber Lid

NOTE

For optimal performance, take care to keep the Hyb

Chamber steady and level when lifting or moving. Avoid shaking the Hyb Chamber, keep the Hyb Chamber parallel to the lab bench while you transfer it to the Illumina

Hybridization Oven.

4. Place the Hyb Chamber into the 48°C Illumina Hybridization Oven so that the clamps of the Hyb Chamber face the left and right side of the oven. The Illumina logo on top of the Hyb Chamber should be facing you.

Figure 43 Hyb Chamber Correctly Placed in Hyb Oven

5. If you are loading multiple Hyb Chambers, stack them on top of each other. You can stack up to 4 Hyb Chambers.

Infinium II Assay Two-Sample HC Protocols

46 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

NOTE

If you are stacking multiple Hyb Chambers in the Illumina

Hybridization Oven, make sure the feet of the top Hyb

Chamber fit into the matching indents on top of the bottom Hyb Chamber. This will hold the Hyb Chambers in place while they are rocking.

Figure 44 Two Hyb Chambers Correctly Placed in Hyb Oven

Part # 11280749 Rev. A

Hybridize HC BC2 47

Figure 45 Incorrectly Placed Hyb Chamber

6. Start the rocker (optional).

7. Incubate at 48°C for at least 16 hours but no more than 24 hours.

8. On the lab tracking worksheet

,

enter the start and stop times.

9. Place RA1 into the freezer at -20°C for use the next day.

Resuspend XC4 Reagent for XStain BC2

Keep the XC4 in the bottle in which it was shipped until ready for use. In preparation for the XStain protocol, follow these steps to resuspend the XC4 reagent:

1. Add 330 ml 100% EtOH to the XC4 bottle. The final volume will be

350 ml.

Each XC4 bottle (350 ml) has enough solution to process up to 24 Bead-

Chips.

2. Shake vigorously for 15 seconds.

3. Leave the bottle upright on the lab bench overnight.

NOTE

If the XC4 was not left to resuspend overnight, you can still proceed with the assay. Add the EtOH and put the XC4 on its side on a rocker to resuspend. Leave it there until the

BeadChips are ready for coating.

4. Shake again to ensure that the pellet is completely resuspended. If any coating is visible, vortex at 1625 rpm until it is in complete suspension.

Infinium II Assay Two-Sample HC Protocols

48 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Once resuspended with 330 ml 100% EtOH, use XC4 at room temperature. You can store it at 4°C overnight, but thaw it again before use.

5. Proceed to Wash BC2 on page 49.

Part # 11280749 Rev. A

Wash BC2 49

Wash BC2

In this process you prepare the BeadChips for the XStain BC2 process. First, you remove the coverseals from the BeadChips and wash the BeadChips in

WB1 reagent followed by PB1 reagent. Next, you assemble the BeadChips into the Flow-Through Chambers under PB1 buffer.

Figure 46 Washing BeadChip

Estimated Time

Hands-on time: ~20 minutes for 4 BeadChips (8 samples)

Consumables

Item

PB1

WB1

Multi-Sample BeadChip

Alignment Fixture

Te-Flow Flow-Through

Chambers (with Black Frames,

Spacers, Glass Back Plates, and Clamps)

4

Wash Dish 2

Bottle

1

Wash Rack 1

Quantity

(per 4 BeadChips)

350 ml

Storage Supplied By

Room temperature

-20°C

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

Infinium II Assay Two-Sample HC Protocols

50 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

This protocol involves the use of an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. Refer to the Infinium Assay

MSDS on your Documentation CD for complete information.

Preparation

`

Thaw WB1 to room temperature (22°C) and allow to equilibrate. Ensure that the solution is completely redissolved.

`

Fill a wash dish with 200 ml WB1. Label the dish “WB1”.

`

Fill a wash dish with 200 ml PB1. Label the dish “PB1”.

`

Fill the BeadChip Alignment Fixture with 150 ml PB1.

`

Separate the clear plastic spacers from the white backs.

`

Clean the glass back plates as directed in the

Infinium II Assay Lab Setup and Procedures Guide

.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• WB1 bottle barcode

• PB1 bottle barcode

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Steps Wash BC2

1. Attach the wire handle to the rack and submerge the wash rack in the wash dish containing 200 ml WB1 (Figure 47).

2. Remove Hyb Chamber inserts from the Hyb Chambers. Remove

BeadChips from the Hyb Chamber inserts one at a time.

Part # 11280749 Rev. A

Wash BC2 51

Figure 47 Wash Rack in Wash Dish Containing WB1

3. Remove the coverseal from each BeadChip (Figure 48): a. Using powder-free gloved hands, hold the BeadChip in one hand between thumb and forefinger, with the front side of the BeadChip facing away from you.

b. Remove the coverseal by stripping it off in a downward direction, starting with the barcode end.

c.

Discard the seal.

CAUTION

To ensure no solution splatters on you, Illumina recommends removing the coverseal over an absorbent cloth or paper towels, preferably in a hood.

Figure 48 Removing the Coverseal

CAUTION

Do not touch the arrays!

4. Immediately and carefully slide each BeadChip into the wash rack one at a time, making sure that the BeadChip is completely submerged in the

WB1 (Figure 49).

Infinium II Assay Two-Sample HC Protocols

52 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Figure 49 Placing BeadChips in Wash Dish Containing WB1

5. Repeat steps 3 through 5 for each BeadChip.

6. Once all the BeadChips are in the wash rack, move the wash rack up and down for 1 minute, breaking the surface of the WB1 with gentle, slow agitation.

7. Once all BeadChips have been individually washed in WB1, remove the wash rack from the wash dish and place it immediately into the wash dish containing PB1. Make sure that the BeadChips are completely submerged.

8. Move the wash rack up and down for 1 minute, breaking the surface of the PB1 with gentle, slow agitation.

9. Immediately wash the Hyb Chamber reservoirs with dH

2

O and scrub them clean with a small brush, ensuring that no PB2 remains.

CAUTION

It is important to wash the reservoirs immediately and thoroughly to ensure that no traces of PB2 remain in the wells.

Part # 11280749 Rev. A

Wash BC2 53

Assemble Flow-Through Chamber

1. For each BeadChip, place a black frame into the Multi-Sample BeadChip

Alignment Fixture (Figure 50), which is pre-filled with PB1.

Figure 50 Placing Black Frames into Alignment Fixture

2. Place each BeadChip into a black frame. Align the barcode with the

ridges stamped onto the Alignment Fixture (Figure 51).

Figure 51 Placing BeadChip into Black Frame on Alignment Fixture

3. Place a clear spacer onto the top of each BeadChip (Figure 52). Use the

Alignment Fixture grooves to guide the spacers into proper position.

Be sure to use the clear plastic spacers, not the white ones.

NOTE

Infinium II Assay Two-Sample HC Protocols

54 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Groove for

Positioning Spacer

White Spacer

Do Not Use

Clear Spacer

Figure 52 Placing Clear Plastic Spacer onto BeadChip

4. Place the Alignment Bar onto the Alignment Fixture (Figure 53). Fit the

groove in the Alignment Bar over the metal tab on the Alignment Fixture.

Figure 53 Placing Alignment Bar onto Alignment Fixture

5. Use a Whoosh duster or laboratory air gun to quickly remove any accumulated dust from the glass back plates just before placing them onto the BeadChips.

6. Place a clean glass back plate on top of the clear spacer covering each

BeadChip. The plate reservoir should be at the barcode end of the

Part # 11280749 Rev. A

Wash BC2 55

BeadChip, facing inward to create a reservoir against the BeadChip

surface (Figure 54).

Reservoir at Barcode End of Glass Back Plate

Glass Back Plate in Position

Figure 54 Placing Glass Back Plate onto BeadChip

7. Attach the metal clamps as follows (Figure 55):

a. Gently push the glass back plate up against the Alignment Bar with one finger.

b. Place the first metal clamp around the Flow-Through Chamber so that one stripe shows between it and the Alignment Bar.

c.

Place the second metal clamp around the Flow-Through Chamber at the barcode end, just below the reagent reservoir, so that no stripes show between the clamp and the barcode.

Infinium II Assay Two-Sample HC Protocols

56 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

One Stripe Shows

Between First Clamp and Alignment Bar

Glass Back Plate

Pressed Against

Alignment Bar

No Stripes Show

Between Second

Clamp and Barcode

Figure 55 Securing Flow-Through Chamber Assembly with Metal Clamps

8. With scissors, trim the spacer at the non-barcode end of the assembly

(Figure 56). Slip the scissors up over the barcode to trim the other end.

Trim Spacer at Non-Barcode

End of Flow-Through Chamber

Trim Spacer at Barcode End of

Flow-Through Chamber

Figure 56 Trimming Spacer Ends from Flow-Through Chamber Assembly

9. Discard unused reagents in accordance with facility standards.

10. Proceed to Single-Base Extension and Stain BC2 on page 57.

Part # 11280749 Rev. A

Single-Base Extension and Stain BC2 57

Single-Base Extension and Stain BC2

In this process, RA1 reagent is used to wash away unhybridized and nonspecifically hybridized DNA sample. TEM reagent is dispensed into the Flow-

Through Chambers to extend primers hybridized to DNA on the BeadChip.

This reaction incorporates labeled nucleotides into the extended primers.

95% formamide/1 mM EDTA is then added to remove the hybridized DNA.

After neutralization using the XC3 reagent, the labeled, extended primers undergo a multi-layer staining process on the Chamber Rack. Finally, the

BeadChips are removed from the Flow-Through Chambers, washed in the

PB1 reagent, coated with XC4, and dried.

(g DN A )

A

T*

C*

G

(g DN A )

* St ain in red channel

* St ain in green channel

Figure 57 Extending and Staining BeadChip

Estimated Time

Hands-on time: ~3 hours for 4–24 BeadChips

Total time: ~4 hours for 4 BeadChips

Consumables

XC1

XC2

TEM

XC3

Item

RA1

LTM (Make sure that all LTM tubes indicate the same stain temperature on the label)

1 tube

ATM 1 tube

PB1 Bottle (310 ml)

XC4

Alconox Powder Detergent

Quantity

(Per 4 BeadChips)

Storage

10 ml (see Setup for special instructions)

-20°C

1 tube -20°C

1 tube

1 tube

49 ml

-20°C

-20°C

Room temperature

-20°C

Bottle (310 ml) as needed

-20°C

Room temperature

-20°C

Supplied By

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

Infinium II Assay Two-Sample HC Protocols

58 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Item

EtOH

95% formamide/1 mM EDTA

Quantity

(Per 4 BeadChips) as needed

Storage

15 ml (per 4

BeadChips)

Supplied By

Room temperature

-20°C

Illumina

User

This protocol involves the use of an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. Refer to the Infinium Assay

MSDS on your Documentation CD for complete information.

Preparation

`

RA1 is shipped and stored at -20°C. Gradually warm the RA1 reagent to room temperature (22°C), preferably in a 20–25°C water bath. Gently mix to dissolve any crystals that may be present.

`

Place all reagent tubes in a rack in the order in which they will be used

(Figure 58). If frozen, allow them to thaw to room temperature (22°C) and

centrifuge to 3000 xg for 3 minutes.

TEM

95% Formamide / 1 mM EDTA

XC2

LTM

XC1

ATM

XC3

PB1

RA1

XC4

Figure 58 XStain BC2 Reagent Tubes and Bottles

`

Dispense all bottled reagents into disposable reservoirs, as they are needed.

`

On the lab tracking worksheet, record:

• Date/Time

Part # 11280749 Rev. A

Single-Base Extension and Stain BC2 59

• Operator

• RA1 barcode

• XC3 barcode

• XC1 barcode(s)

• XC2 barcode(s)

• TEM barcode(s)

• LTM barcode(s)

• ATM barcode(s)

• PB1 barcode

• XC4 barcode(s)

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Set Up the Chamber Rack

1. Ensure the water circulator reservoir is filled with water to the appropriate level. See the

VWR Operator’s Manual,

VWR part # 110-229.

2. Turn on the water circulator and set it to a temperature that brings the

Chamber Rack to 44°C at equilibrium.

This temperature may vary depending on facility ambient conditions.

Infinium II Assay Two-Sample HC Protocols

60 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

(Blowup)

Water Circulator with

Programmable Temperature

Control

Reservoir Cover

Chamber Rack on Robot Bed

Figure 59 Water Circulator Connected to Chamber Rack

3. The temperature displayed on the water circulator LCD screen may differ from the actual temperature on the Chamber Rack. Confirm this using the temperature probe for the Chamber Rack.

4. Remove the bubbles trapped in the Chamber Rack. You must do this every time you run this process. Follow instructions in the

Te-Flow (Tecan

Flow-Through Module) Operating Manual

, Tecan Doc ID 391584.

5. Use the Illumina Temperature Probe in several locations to ensure that the Chamber Rack is at 44°C (Figure 60).

Do not leave the temperature probe in the first three rows of the Chamber Rack. Reserve this space for BeadChips.

Part # 11280749 Rev. A

Single-Base Extension and Stain BC2 61

Temperature Probe

Temperature Probe in Chamber Rack

Figure 60 Illumina Temperature Probe in Chamber Rack

6. For accurate temperature measurement, ensure the Temperature Probe is touching the base of the Chamber Rack.

Steps

This protocol includes the following steps:

`

Single-Base Extension on page 61

`

Stain BeadChip on page 62

`

Preparing Wash Dishes and Tube Racks on page 63

`

Wash and Coat 8 BeadChips on page 63

CAUTION

The remaining steps must be performed without interruption.

Single-Base Extension

1. When the Chamber Rack reaches 44°C, quickly place each Flow-Through

Chamber assembly into the Chamber Rack.

For 4 BeadChips, place the Flow-Through Chambers in every other position, starting at 1, in the first row of the Chamber Rack. For larger numbers of BeadChips, fill all positions in the first row, then the second and third.

2. Ensure each Flow-Through Chamber is properly seated on its rack to allow adequate heat exchange between the rack and the chamber.

3. On the lab tracking worksheet, record the Chamber Rack position for each BeadChip.

4. Into the reservoir of each Flow-Through Chamber, dispense:

Infinium II Assay Two-Sample HC Protocols

62 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

a. 150 μl RA1 (Figure 61). Incubate for 30 seconds. Repeat 5 times.

Pipette tip must not contact

BeadChip surface

Figure 61 Dispensing RA1 into Each Flow-Through Chamber

CAUTION

Do not allow pipette tips to contact BeadChip surface.

Touch off in the reservoir of the glass back plate.

b. 450 μl XC1. Incubate for 10 minutes.

c.

450 μl XC2. Incubate for 10 minutes.

d. 200 μl TEM. Incubate for 15 minutes.

e. 450 μl 95% formamide/1 mM EDTA. Incubate for 1 minute. Repeat once.

f.

Incubate 5 minutes.

g. Begin ramping the Chamber Rack temperature to the temperature indicated on the LTM tube, or to 37°C if none is shown.

h. 450 μl XC3. Incubate for 1 minute. Repeat once.

5. Wait until the Chamber Rack reaches the correct temperature.

Stain BeadChip

1. If you plan to image the BeadChip immediately after the staining process, turn on the Illumina BeadArray™ Reader now to allow the lasers to stabilize.

2. Into the reservoir of each Flow-Through Chamber, dispense: a. 250 μl LTM and incubate for 10 minutes.

b. 450 μl XC3 and incubate for 1 minute. Repeat once, and then wait

5 minutes.

c.

250 μl ATM and incubate for 10 minutes.

d. 450 μl XC3 and incubate for 1 minute. Repeat once, and then wait

5 minutes.

Part # 11280749 Rev. A

Single-Base Extension and Stain BC2 63 e. 250 μl LTM and incubate for 10 minutes.

f.

450 μl XC3 and incubate for 1 minute. Repeat once, and then wait

5 minutes.

g. 250 μl ATM and incubate for 10 minutes.

h. 450 μl XC3 and incubate for 1 minute. Repeat once, and then wait

5 minutes.

i.

250 μl LTM and incubate for 10 minutes.

j.

450 μl XC3 and incubate for 1 minute. Repeat once, and then wait

5 minutes.

3. Incubate 5 minutes.

4. Immediately remove the Flow-Through Chambers from the Chamber

Rack and place horizontally on a lab bench at room temperature (22°C).

Preparing Wash Dishes and Tube Racks

Before starting the Wash and Coat process, please read these important notes:

• Take the utmost care to minimize the chance of lint or dust entering the wash dishes, which could transfer to the BeadChips. Place wash dish covers on wash dishes when stored or not in use. Clean wash dishes with low-pressure air to remove particulates prior to use.

• In preparation for XC4 BeadChip coating, wash tube racks and wash dishes thoroughly before and after use. Rinse with DI water.

Immediately following wash, place racks and wash dishes upside down on a wash rack to dry.

• Place Kimwipes in three layers on the lab bench. Place a tube rack on top of these Kimwipe layers. Do not place on absorbent lab diapers.

You will place the staining rack containing BeadChips on this tube rack after removing it from the XC4 wash dish.

• Prepare an additional clean tube rack (Illumina-provided from VWR catalog # 60916-748) that fits the internal dimensions of vacuum desiccator for removal of the BeadChips. Allow one rack per

8 BeadChips. No Kimwipes are required under this tube rack.

Wash and Coat 8 BeadChips

1. Lay out the following equipment on the lab bench:

• 1 staining rack

• 1 vacuum desiccator

• 1 tube rack

• Self-locking tweezers

• Large Kimwipes

• Vacuum hose

1. Set up two top-loading wash dishes, labeled as shown (Figure 62).

2. To indicate the fill volume before filling wash dishes with PB1 and XC4, pour 310 ml water into the wash dishes and mark the water level on the side. Empty the water from the wash dish. This enables you to pour

Infinium II Assay Two-Sample HC Protocols

64 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol reagent directly from the PB1 and XC4 bottles into the wash dishes, minimizing contaminant transfer from labware to wash dishes.

Wash Dishes

Staining Rack

Figure 62 PB1 and XC4 Wash Dishes with Staining Rack

3. Pour 310 ml PB1 into the wash dish labeled “PB1.”

4. Submerge the unloaded staining rack into the wash dish with the locking arms facing you. This orients the staining rack so that you can safely remove the BeadChips.

Locking Arms

Tab

Figure 63 Staining Rack Locking Arms and Tabs

CAUTION

If the staining rack handle is not correctly oriented, the

BeadChips may be damaged when you remove the staining rack handle before removing the BeadChips.

Let the staining rack sit in the wash dish. You will use it to carry the Bead-

Chips after disassembling the Flow-Through Chambers.

5. One at a time, disassemble each Flow-Through Chamber:

a. Use the dismantling tool to remove the two metal clamps (Figure 64).

Part # 11280749 Rev. A

Single-Base Extension and Stain BC2 65

CAUTION

It is important to use the dismantling tool to avoid chipping the glass back plates.

Figure 64 Removing Metal Clamps from Flow-Through Chamber

b. Remove the glass back plate.

c.

Set the glass back plate aside. When you finish the XStain BC2 protocol, clean the glass back plates as described in the Infinium II

Assay Lab Setup and Procedures Guide.

d. Remove the spacer. To avoid damaging the stripes on the BeadChip, pull the spacer out so that the long sides slide along the sides of the

BeadChip.

e. Remove the BeadChip.

CAUTION

Do not touch the face of the BeadChips. Handle them by the barcode end or by the edges.

6. Place BeadChips in the staining rack while it is submerged in PB1. Put four BeadChips above the staining rack handle and four below.

If necessary, briefly lift the staining rack out of the wash dish to seat the

BeadChip. Replace it immediately after inserting the BeadChip.

7. Ensure that the BeadChip barcodes are correctly positioned in the staining rack, with the labels facing away from you. This is essential for proper handling and coating.

CAUTION

Do not allow the BeadChips to dry. Submerge each

BeadChip in the wash dish as soon as possible.

8. Ensure that the BeadChips are completely submerged.

9. Move the staining rack up and down 10 times, breaking the surface of the PB1.

Infinium II Assay Two-Sample HC Protocols

66 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Figure 65 Washing BeadChips in PB1

NOTE

If the top edges of the BeadChips begin to touch during either PB1 or XC4 washes, gently move the staining rack back and forth to separate the slides. It is important for the solution to circulate freely between all BeadChips.

10. Allow the BeadChips to soak for an additional 5 minutes.

CAUTION

Do not leave the BeadChips submerged in PB1 for longer than 30 minutes.

11. Shake the XC4 bottle vigorously to ensure complete resuspension. If necessary, vortex until completely dissolved.

12. Pour 310 ml XC4 into the dish labeled “XC4,” and cover the dish to prevent any lint or dust from falling into the solution.

Use the XC4 within 10 minutes after filling the wash dish.

NOTE

13. Remove the staining rack from the PB1 dish and place it directly into the

wash dish containing XC4 (Figure 66). The barcode labels on the

BeadChips must face away from you, while the locking arms on the handle face towards you, for proper handling and coating.

Part # 11280749 Rev. A

Single-Base Extension and Stain BC2 67

Figure 66 Moving BeadChips from PB1 to XC4

14. Move the staining rack up and down 10 times, breaking the surface of the XC4.

NOTE

If the top edges of the BeadChips begin to touch during either PB1 or XC4 washes, gently move the staining rack back and forth to separate the slides. It is important for the solution to circulate freely between all BeadChips.

15. Allow the BeadChips to soak for an additional 5 minutes.

CAUTION

Use XC4 only once. To process subsequent BeadChips, use a new, clean wash dish with fresh XC4.

16. Prepare a clean tube rack for the staining rack by placing two folded

Kimwipes under the tube rack.

17. Prepare one additional tube rack per 8 BeadChips (Illumina-provided from VWR catalog # 60916-748) that fits the internal dimensions of vacuum desiccator.

18. Remove the staining rack in one smooth, rapid motion and place it directly on prepared tube rack, making sure the barcodes face up and

the locking arms and tab face down (Figure 67).

Infinium II Assay Two-Sample HC Protocols

68 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Figure 67 Staining Rack in Correct Orientation

19. To ensure uniform coating, place the staining rack on the center of the tube rack, avoiding the raised edges.

Figure 68 Moving the Staining Rack from XC4 to Tube Rack

20. For the top four BeadChips, working top to bottom: a. Continuing to hold the staining rack handle, carefully grip each

BeadChip at its barcode end with self-locking tweezers.

NOTE

The XC4 coat is slippery and makes the BeadChips difficult to hold. The self-locking tweezers grip the BeadChip firmly and help prevent damage.

b. Place the BeadChip on a tube rack with the barcode facing up and

towards you (Figure 70).

21. Holding the top of the staining rack in position, gently remove the staining rack handle by grasping the handle between the thumb and

Part # 11280749 Rev. A

Single-Base Extension and Stain BC2 69 forefinger. Push the tab up with your thumb and push the handle away from you (unlocking the handle), then pull up the handle and remove

(Figure 69).

Tab

Handle

Figure 69 Removing Staining Rack Handle

22. Remove the remaining BeadChips to the tube rack as shown in Figure

70, with six BeadChips on top of the rack and two BeadChips on the

bottom. The barcode ends should be towards you, and the BeadChips should be completely horizontal.

Figure 70 Placing BeadChips on Tube Rack

CAUTION

To prevent wicking and uneven drying, do not allow the

BeadChips to rest on the edge of the tube rack or to touch each other while drying.

Infinium II Assay Two-Sample HC Protocols

70 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

23. Place the tube rack in the vacuum desiccator. Each dessicator can hold

one tube rack (8 BeadChips) (Figure 71).

24. Remove the red plug from the three-way valve before applying vacuum pressure.

25. Start the vacuum, using at least 508 mm Hg (0.68 bar).

26. To ensure that the dessicator is properly sealed, gently lift the lid of the vacuum desiccator. It should not lift off the desiccator base.

Figure 71 Testing Vacuum Seal

27. Dry under vacuum for 50–55 minutes.

Drying times may vary according to room temperature and humidity.

28. Release the vacuum by turning the handle very slowly.

Air should enter the desiccator very slowly to avoid disturbing the contents. Improper use of the vacuum desiccator can result in damage to the BeadChips. This is especially true if you remove the valve plug while a vacuum is applied. For detailed vacuum desiccator instructions, see the documentation included with the desiccator.

29. Store desiccator with the red valve plug in the desiccator’s three-way valve to stop accumulation of dust and lint within the valve port.

30. Touch the borders of the chips (do not touch the stripes) to ensure that the etched, bar-coded side of the BeadChips are dry to the touch.

31. If the underside feels tacky, manually clean the underside of the

BeadChip to remove any excess XC4. The bottom two BeadChips are the most likely to have some excess.

a. Hold the BeadChip at a downward angle to prevent excess EtOH from dripping from the wipe onto the stripes.

Part # 11280749 Rev. A

Image BC2 71 ti a. Wrap a pre-saturated Prostat EtOH Wipe around your index finger.

b. Wipe along the underside of the BeadChip five or six times, until the surface is clean and smooth.

Do not touch the stripes.

CAUTION

32. Clean the glass back plates. For instructions, see the Infinium II Assay Lab

Setup and Procedures Guide.

33. Clean the Hyb Chambers: a. Remove the rubber gaskets from the Hyb Chambers.

b. Rinse all Hyb Chamber components with DI water.

c.

Thoroughly rinse the humidifying buffer reservoirs.

34. Discard unused reagents in accordance with facility standards.

35. Do one of the following:

•

Proceed to Image BC2 on page 71.

• Store the BeadChips in the Illumina BeadChip Slide Storage Box inside a vacuum desiccator at room temperature (22°C). Image the

BeadChips within 72 hours.

Image BC2

The BeadChips are now ready for scanning. For instructions, see the Infinium

II Assay Lab Setup and Procedures Guide. Image the BeadChips within

72 hours.

Infinium II Assay Two-Sample HC Protocols

72 CHAPTER 2

Two-Sample HC BeadChip Manual Protocol

Part # 11280749 Rev. A

Chapter 3

Two-Sample HC BeadChip

Automated Protocol

115

129

138

163

99

101

104

111

80

85

87

94

74

74

76

78

Topics

Introduction

Two-Sample HC BeadChip Automated Workflow

Equipment, Materials, and Reagents

Make Standard DNA Plate (Optional/Infinium LIMS)

Make Quant (Optional/LIMS)

Read Quant (Optional/LIMS)

Make the AMP2 Plate

Make Multiple AMP2 Plates

Incubate the AMP2 Plate

Fragment the AMP2 Plate

Precipitate the AMP2 Plate

Resuspend the AMP2 Plate

Hybridize HC BC2

Wash BC2

Single-Base Extension & Stain BC2

Image BC2

Infinium II Assay Two-Sample HC Protocols 73

74 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Introduction

This chapter describes pre- and post-amplification automated laboratory protocols for the Two-Sample HC BeadChip. Follow the protocols in the order shown.

Some of the tasks in this chapter make reference to Infinium LIMS (Laboratory

Information Management System). If you are not running Infinium LIMS, disregard those instructions. For information about how to use Infinium LIMS, see the Infinium II LIMS User Guide.

Two-Sample HC BeadChip Automated Workflow

Figure 72 graphically represents the Illumina Infinium II Assay automated

workflow, with or without Infinium LIMS, for two-sample Genotyping

BeadChips. These protocols describe the procedure for preparing eight DNA samples. To process 48 or 96 samples, scale up the protocols accordingly.

Part # 11280749 Rev. A

Day 1

Make Quant

(Optional/LIMS)

Robot: 25 min/8 samples

Reagents

Lambda DNA

PicoGreen dsDNA

1X TE

Output

QNT Plate

Standard QNT Plate

Read Quant

(Optional/LIMS)

Hands-on: ~5 min/plate

Output

Electronic File with

Information about

Sample DNA

Day 2

Fragment AMP2

Robot: 10 min/8 samples

Incubation: 60 min

Reagents

FRG

Output

AMP2 Plate

Precip AMP2

Robot: 20 min/8 samples

Incubation: 55 min

Dry: 60 min

Reagents

2-propanol

PA1

Output

AMP2 Plate

Make Multi AMP2

Robot:

35 min/24 samples

Reagents

0.1N NaOH

MP1

AMM

Output

1–4 AMP2 Plates

Make AMP2

Robot: 20 min/8 samples

Reagents

0.1N NaOH

MP1

AMM

Output

AMP2 Plate

Resuspend AMP2

Robot: 5 min/plate

Incubation: 1 hour

Reagents

RA1

Output

AMP2 Plate

Introduction 75

Day 3

Wash BC2

Robot: 20 min/4

BeadChips

Reagents

WB1

PB1

Output

BeadChip

XStain BC2

Robot:

130 min/8 samples

Dry: 1 hour

Reagents

RA1

95% Formamide /

1 mM EDTA

PB1

XC1

XC2

XC3

XC4

TEM

LTM

ATM

Output

BeadChip

Image BC2

Scan: 45 min/BeadChip

Output

Image and Data Files

Incubate AMP2

Incubation: 20–24 hours

Output

AMP2 Plate with

Amplified DNA

Hyb HC BC2

Robot: 10 min/8 samples

Incubation: 16–24 hours

Reagents

PB2

Output

BeadChip

Pre-Amp

Post-Amp

Optional stop, cold storage

Overnight incubation

Figure 72 Two-Sample HC BeadChip Automated Workflow

Infinium II Assay Two-Sample HC Protocols

76 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Equipment, Materials, and Reagents

These items are specifically required for the two-sample, automated

BeadChip assay. For a list of other equipment, materials, and reagents needed in an Infinium II Assay lab, see the Infinium II Assay Lab Setup and

Procedures Guide.

Equipment User-Supplied

Table 8 User-Supplied Equipment

Item

Vacuum desiccator (1 per 8 BeadChips processed simultaneously)

Vacuum tubing

2 Tecan eight-tip robots (one for pre- and one for post-amplification processes)

Source

VWR Catalog # 24988-197

VWR Catalog # 62995-335

Non-LIMS customers

• SC-30-401 (110V) - North America and

Japan

• SC-30-402 (220V) - EU and Asia Pacific

(Except Japan)

Infinium LIMS customers

• SC-30-403 (110V) - North America and

Japan

• SC-30-404 (220V) - EU and Asia Pacific

(Except Japan)

Forceps VWR International, Catalog # 25601-008

Auto-desiccator cabinet (Optional—allows scanning of BeadChips up to three days after processing)

VWR International, Catalog # 74950-342

Illumina-Supplied

Table 9 Illumina-Supplied Materials

Item

Multi-Sample BeadChip Alignment Fixture

Robot BeadChip Alignment Fixture (6)

Materials User-Supplied

Table 10 User-Supplied Materials

Item

96-well, black, flat-bottom Fluotrac 200 plates

Catalog or Part #

Part # 218528

Part # 222691

Source

Greiner, catalog # 655076

Part # 11280749 Rev. A

Equipment, Materials, and Reagents 77

Table 10 User-Supplied Materials

Item

Foil adhesive seals (Microseal “F”)

Aluminum foil

Reservoir, full, 150 ml

Reservoir, half, 75 ml

Reservoir, quarter, 40 ml

Reservoir frame

Tube racks for vacuum desiccator (1 for every 8 BeadChips to be processed simultaneously; must fit internal dimensions of vacuum desiccator)

Vacuum source (greater than 508 mm Hg (0.68 bar)

(Recommended) Vacuum gauge for vacuum desiccator

Source

MJ Research, Catalog # MSF-1001

Beckman Coulter, catalog # 372784

Beckman Coulter, catalog # 372786

Beckman Coulter, catalog # 372790

Beckman Coulter, catalog # 372795

VWR catalog # 66023-526

Illumina-Supplied

`

AMP2 barcode labels

`

WG#-DNA barcode labels

`

QNT barcode labels

Reagents Illumina-Supplied

Table 11 Illumina-Supplied Reagents

Item

MP1—Neutralization solution

AMM—Amplification Master Mix

FRG—Fragmentation solution

PA1—Precipitation solution

RA1—Resuspension, hybridization, and wash solution

PB1—Reagent used to prepare BeadChips for hybridization

PB2—Humidifying buffer used during hybridization

XC1—XStain BeadChip solution 1

XC2—XStain BeadChip solution 2

TEM—Two-Color Extension Master Mix

Part #

11190751

11192044

11190022

11190031

11191914

11191922

11191130

11208288

11208296

11208309

Infinium II Assay Two-Sample HC Protocols

78 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Table 11 Illumina-Supplied Reagents

Item

XC3 (80 ml)—XStain BeadChip solution 3

XC3 (240 ml)—XStain BeadChip solution 3

LTM—Labeling Two-Color Master Mix

ATM—Anti-Stain Two-Color Master Mix

XC4—XStain BeadChip solution 4

Part #

11208392

11208421

11208325

11208317

11208430

Make Standard DNA Plate (Optional/Infinium LIMS)

This process creates a Standard DNA plate with specific concentrations of

DNA in the wells. Use this plate as input into the Make Quant process. If your

DNA has already been quantified and you are not running Infinium LIMS

(Laboratory Information Management System), you do not have to perform this step.

For information about how to use Infinium LIMS, see the

Infinium II LIMS User

Guide

.

Consumables

Item Quantity Storage

Lambda DNA

1X TE

See formula below

(10 mM Tris-HCl pH8.0, 1 mM EDTA)

Room temperature

96-well microtiter plate (MIDI) 1 per 96 samples

Supplied By

User

User

User

Preparation

`

Remove PicoGreen reagent from freezer and thaw at room temperature for 60 minutes in a light-impermeable container.

`

Label a 96-well 0.65 ml MIDI plate “Standard QNT.”

Steps Make Standard QNT MIDI Plate

In this process, you create a Standard QNT plate with serial dilutions of stock

Lambda DNA in the wells of column 1.

1. Place stock Lambda DNA in well A1 of the Standard QNT MIDI plate and dilute it to 75 ng/μl in a final volume of 233.3 μl.

a. Use the following formula to calculate the amount of stock Lambda

DNA to add to A1:

Part # 11280749 Rev. A

Make Standard DNA Plate (Optional/Infinium LIMS) 79

(233.3

μl

) X (75 ng/μl)

(stock Lambda DNA concentration)

= μl

of stock

Lambda

DNA to add to A1 b. Dilute the stock DNA in well A1 using the following formula:

μl of 1X TE to add to A1 =

233.3

μ well A1 l -

μl

of stock

Lambda

DNA in

2. Add 66.7 μl 1X TE to well B of column 1 of the same plate.

3. Add 100 μl 1X TE to wells C, D, E, F, G, and H of column 1 of the same plate.

λ

DNA 1X TE

75 ng/µL

66.7

µL

100

µL

100

µL

100

µL

100

µL

100

µL

100

µL

A B C D E F G H

Figure 73 MIDI Plate Wells

MIDI Plate

4. Pipette the contents of A1 up and down 10 times to mix.

5. Transfer 133.3 μl of Lambda DNA from well A1 into well B1, and then pipette the contents of well B1 up and down 10 times.

6. Change pipette tips. Transfer 100 μl from well B1 into well C1, and then pipette the contents of well C1 up and down 10 times.

7. Change pipette tips. Transfer 100 μl from well C1 into well D1, and then pipette the contents of well D1 up and down 10 times.

8. Change pipette tips. Transfer 100 μl from well D1 into well E1, and then pipette the contents of well E1 up and down 10 times.

9. Change pipette tips. Transfer 100 μl from well E1 into well F1, and then pipette mix the contents of well F1 up and down 10 times.

10. Change pipette tips. Transfer 100 μl from well F1 into well G1, and then pipette the contents of well G1 up and down 10 times.

11. Do not transfer solution from well G1 to well H1. Well H1 serves as the blank 0 ng/μl Lambda DNA.

Infinium II Assay Two-Sample HC Protocols

80 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Table 12 Concentration of Lambda DNA Standards

Row-Column

A1

B1

C1

D1

E1

F1

G1

H1

Conc. (ng/μl)

75

50

25

12.5

6.25

3.125

1.5625

0

Final Volume in well (μl)

100

100

100

100

100

100

200

100

12. Cover the plate with a cap mat.

13. Do one of the following:

• Store the plate at 4°C for future use.

•

Proceed to Make Quant (Optional/LIMS) on page 80.

Make Quant (Optional/LIMS)

In this process, you create one to three QNT plates for use in the Molecular

Dynamics Fluorometer (if available). The fluorometer quantifies the DNA present in the sample and enters that data in to Infinium LIMS. Quantification ensures that there is enough sample DNA to generate good data. Use this procedure if you are running Infinium LIMS or if you have a fluorometer in your lab. For information about how to use Infinium LIMS, see the Infinium II

LIMS User Guide.

Illumina recommends the Molecular Probes PicoGreen assay for quantitation of dsDNA samples. The PicoGreen assay can quantitate small DNA volumes, and measures DNA directly.

Estimated Time

Robot time:

• 20 minutes per 8 samples (1 plate)

• 40 minutes for 16 samples (2 plates)

• 60 minutes for 24 samples (3 plates; repeat twice for 48 samples, or three times for 96 samples)

Part # 11280749 Rev. A

Make Quant (Optional/LIMS) 81

Consumables

Item Quantity Storage

WG#-DNA plate with 96 DNA samples, normalized to

50 ng/μl

8, 16, 24, 32, 48, or

96 DNA samples in 1 to 3 plates

-20°C

Room temperature

1X TE (10 mM Tris-HCl pH

8.0, 1 mM EDTA (TE))

PicoGreen dsDNA

Black Fluotrac plate

Standard DNA plate

1 plate for each

WG#-DNA plate

1 plate for each

Standard DNA plate

-20°C

1 plate -20°C

Supplied By

User

User

User

Illumina

Illumina

NOTE

PicoGreen is susceptible to differential contaminants. False positives may occur for whole-genome amplification.

Therefore, it is important to quantify the input into the whole-genome amplification reaction.

Preparation

`

For each WG#-DNA plate you plan to quantify, place a QNT barcode label on a new black Fluotrac plate. Position the label on the skirt of the plate, where the manufacturer’s name appears.

`

If you are using Infinium LIMS, make sure the WG#-DNA plates have been accessioned.

`

Remove the WG#-DNA plate(s) from the refrigerator or freezer. If frozen, thaw completely.

`

In the Sample Sheet, enter the Sample_Name (optional) and

Sample_Plate for each Sample_Well.

Prepare the Robot

For instructions on preparing the robot for use in a protocol, see the Infinium

II Assay Lab Setup and Procedures Guide.

Refer to Figure 81 throughout this protocol. Note that all of the barcodes

face to the right.

Infinium II Assay Two-Sample HC Protocols

82 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

PicoGreen in Half

Reservoir

Standard QNT Plate (Fluotrac)

Standard DNA Plate (MIDI)

WG#-DNA Plate (MIDI)

QNT Plate (Fluotrac)

Figure 74 Tecan Eight-Tip Robot (Make Quant Setup)

Steps Set up the Robot

1. At the robot PC, select DNA Quant | Make Quant.

2. In the DNA Plate Selection dialog box, select the plate type.

Do not mix plate types on the robot bed.

NOTE

Roll the mouse pointer over each picture to see a description of the plate. They should all be MIDI plates or all be TCY plates.

3. (Non-Infinium LIMS only) In the Basic Run Parameters pane, set the value for the Number of DNA/QNT plates (1, 2, or 3) and the Number of

DNA samples (8, 16, 24, 32, 48, or 96).

NOTE

If you are using Infinium LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

Part # 11280749 Rev. A

Make Quant (Optional/LIMS) 83

Figure 75 Make Quant Screen

4. Vortex each WG#-DNA plate:

• For MIDI plate(s): 1450 rpm (actual vortex speed) for 1 minute

• For TCY plate(s): 1250 rpm for 1 minute

5. Centrifuge each WG#-DNA plate to 280 xg for 1 minute.

6. Vortex the Standard DNA plate at 1450 rpm for 1 minute.

7. Centrifuge the Standard DNA plate to 280 xg for 1 minute.

8. Place the WG#-DNA, Standard DNA, Standard QNT, and QNT plates on

the robot bed according to the robot bed map (Figure 75). Place well A1

at the upper-left corner of the robot bed carrier. Remove any plate seals.

9. Proceed to Prepare PicoGreen Dilution.

Prepare PicoGreen Dilution

CAUTION

PicoGreen reagent degrades quickly in the presence of light. Do not use glass containers for PicoGreen reagent.

1. Wrap aluminum foil around a sterile plastic container to prevent light penetration.

2. Make a 1:200 dilution of PicoGreen to 1X TE in the foil-wrapped plastic

container (Table 13). Mix thoroughly.

Infinium II Assay Two-Sample HC Protocols

84 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Table 13 PicoGreen Reagent Volumes

# QNT Plates

1

2

3

PicoGreen Volume (μl)

125

230

325

1X TE Volume (ml)

25

45

65

You can prepare dilutions for up to three sample plates at a time.

The robot transfers stock Lambda DNA dilution into the Standard QNT plate. Stock Lambda DNA is then transferred from the standard DNA plate to the Standard QNT plate.

Start the Robot

1. At the robot PC: a. If you are not running Infinium LIMS, clear the Use Barcodes check box. b. Click Run to start the process. c.

Log in if prompted.

d. Observe the robot start to run to ensure that there are no problems.

The robot transfers sample from the Standard DNA plate to the Standard

Quant plate, and from the WG#-DNA plate(s) to the QNT plate(s).

The robot PC sounds an alert and displays a message when the process is complete.

2. Click OK in the message box.

3. On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• The QNT barcode that corresponds to each WG#-DNA barcode

• The Standard QNT plate that corresponds to each Standard DNA plate.

4. After the robot finishes, immediately: a. Place foil adhesive seals over QNT and Standard QNT plates.

b. Place cap mats on WG#-DNA and Standard DNA plates.

5. Discard unused reagents in accordance with facility requirements.

6. Place the WG#-DNA and Standard DNA plates in the refrigerator (4°C) or freezer (-20°C).

7. Centrifuge the QNT and Standard QNT plates to 280 xg for 1 minute.

8. Proceed to Read Quant (Optional/LIMS) on page 85.

Part # 11280749 Rev. A

Read Quant (Optional/LIMS) 85

Read Quant (Optional/LIMS)

In this process, you use a fluorometer along with the Infinium Fluorometry

Analysis software to interpret the quantified DNA in the QNT plate(s) and obtain the exact concentration of DNA in the sample. This information is saved with the other project data in Infinium LIMS. Illumina recommends using a fluorometer because fluorometry provides DNA-specific quantification. Spectrophotometry may also measure RNA, yielding values that are too high.

For information about how to use Infinium LIMS, see the Infinium II LIMS User

Guide.

Estimated Time

Robot time: 5 minutes per plate

Steps

1. Turn on the fluorometer.

2. Open the Infinium Fluorometry Analysis software.

Figure 76 Infinium Fluorometry Analysis Opening Screen

3. Select Reader Tasks | Read Quant.

4. Select the Use Barcodes check box.

5. Click Read.

6. If prompted, enter your Infinium LIMS user name and password. Click

Login.

7. When asked if you want to read a new Standard QNT plate, click Yes.

Remove the plate seal and load the Standard QNT plate in the open fluorometer tray. Click OK. The fluorometer reads the plate data.

8. Review the data from the Standard QNT plate. Either accept it and go on to the next step, or reject it and read another plate.

Infinium II Assay Two-Sample HC Protocols

86 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

9. Remove the Standard QNT plate from the fluorometer tray.

10. When prompted, enter the number of QNT plates that you want to read.

You can read up to 3 plates. Do not include the Standard QNT plate.

Figure 77 Number of QNT Plates Dialog Box

11. Scan the first QNT barcode, load the plate in the tray, and click OK.

Figure 78 Plate Barcode Dialog Box

The Fluorometry Analysis screen fills in with information about the fluorescence in the wells.

Figure 79 Fluorescence Data

Microsoft Excel opens automatically at the same time and displays the quant data for the QNT plate. There are three tabs in the file:

• SQNT_STD—Plots the RF values against the concentration (ng/μl).

• QNT—Plots the concentration (ng/μl) for each well.

• Data—Compares the data from the Standard QNT plate to the QNT plate you just read.

Part # 11280749 Rev. A

Make the AMP2 Plate 87

The Infinium Fluorometer Analysis software prompts you to indicate whether you wish to use the QNT data shown in the Excel file.

12. Do one of the following:

• Click Yes to send the data to Infinium LIMS. In Infinium LIMS, the

QNT plate moves into the Make AMP2 queue.

• Click No to delete the quant data. You can read the quant data again for the same plate.

13. Scan and read the data for up to two other QNT plates.

14. Discard the QNT plates and reagents in accordance with facility requirements.

15. Proceed to Make the AMP2 Plate on page 87.

Make the AMP2 Plate

This process creates an AMP2 plate for DNA amplification. DNA samples are added to designated well locations, and then 0.1N NaOH is added, denaturing the DNA. Neutralization occurs when the MP1 reagent is added.

Finally, the robot adds AMM (Amplification Master Mix) to the DNA samples.

Figure 80 Denaturing and Neutralizing DNA

Estimated Time

Robot time:

• 20 minutes per 8 samples

• 70 minutes for 48 samples

Consumables

Item

0.1N NaOH

Quantity

15 ml per 8–24 samples

Storage

4°C

WG#-DNA plate with 96 DNA samples (50 ng/μl)

1 plate

MP1

AMM

-20°C

1 tube per 8 samples -20°C

1 tube per 8 samples -20°C

Supplied By

User

User

Illumina

Illumina

Infinium II Assay Two-Sample HC Protocols

88 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Item Quantity

96-well 0.8 ml microtiter plate

(MIDI)

1 plate for up to

24 samples

Storage Supplied By

User

NOTE

Thaw all reagents completely at room temperature (22°C) and allow to equilibrate. Once thawed, gently invert each tube several times to thoroughly mix the reagent. Pulse centrifuge each tube to 280 xg to eliminate bubbles and collect reagent at the bottom of the tube.

Preparation

`

In preparation for Incubate AMP2, preheat the Illumina Hybridization

Oven in the post-amp area to 37°C and allow the temperature to equilibrate.

`

Thaw MP1 and AMM tubes to room temperature (22°C). Gently invert to mix, then pulse centrifuge to 280 xg.

`

Thaw DNA samples to room temperature (22°C).

`

Apply an AMP2 barcode to a new MIDI plate.

`

In the Sample Sheet, enter the Sample_Name (optional) and

Sample_Plate for each Sample_Well.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• WG#-DNA plate barcode

• AMP2 plate barcode

• MP1 tube barcode(s)

• AMM tube barcode(s)

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Prepare the

Robot

For instructions on preparing the robot for use in a protocol, see the Infinium

II Assay Lab Setup and Procedures Guide.

Refer to Figure 81 throughout this protocol. Note that the barcodes all face

to the right.

Part # 11280749 Rev. A

Make the AMP2 Plate 89

MP1 Tube

AMM Tube

AMP2 Plate

NaOH in Quarter

Reservoir

WG#-DNA Plate

(MIDI)

Figure 81 Tecan Eight-Tip Robot (Make AMP2 Setup)

Steps

1. If you do not already have a WG#-DNA plate, dispense DNA into either a:

• MIDI plate: 40 μl to each WG#-DNA plate well

• TCY plate: 30 μl to each WG#-DNA plate well

Apply a barcode label to the new WG#-DNA plate.

2. At the robot PC, select AMP2 Sample Prep Tasks | Make AMP2.

If you want to process multiple AMP2 plates at once, see Make Multiple

AMP2 Plates on page 94.

3. Select the WG#-DNA plate type (MIDI or TCY).

MIDI Plate

TCY Plate

Figure 82 Selecting the DNA Plate Type

Infinium II Assay Two-Sample HC Protocols

90 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Do not mix plate types on the robot bed.

NOTE

4. (Non-Infinium LIMS only) Make sure the Use Barcodes check box is cleared. In the Basic Run Parameters pane, enter the Number of DNA samples (8, 16, or 24) that are in the plate. This value must match the number of DNAs in the plate and the number of DNAs identified in the

DNA manifest.

NOTE

If you are using Infinium LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

Basic Run Parameters

Highlighted Tubes

Robot Bed Map

Figure 83 Make AMP2 Basic Run Parameters

5. Remove the caps. Place the MP1 and AMM tubes in the robot tube rack

according to the robot bed map (Figure 83).

6. Place a quarter reservoir on the robot bed according to the bed map,

and add 15 ml 0.1N NaOH (Figure 83).

7. Place the sealed WG#-DNA plate on the vortexer and secure it with the

provided Velcro straps (Figure 84).

Part # 11280749 Rev. A

Make the AMP2 Plate 91

Figure 84 Securing Plates to Vortexer Platform with Velcro Straps

8. Vortex the sealed WG#-DNA plate at 1600 pm for 1 minute.

9. Centrifuge to 280 xg for 1 minute at 22°C.

10. Place the AMP2 and WG#-DNA plates on the robot bed according to the

bed map (Figure 83). Remove all plate seals.

NOTE

When you remove a cap mat, set it aside, upside down, in a safe location for use later in the protocol. When you place the cap mat back on the plate, be sure to match it to its original plate and orient it correctly.

11. In the lab tracking worksheet, record the plate positions on the robot bed.

12. Make sure that all items are placed properly on the robot bed, that all caps and seals have been removed, and that all the barcodes face to the right.

13. (Non-Infinium LIMS only) At the robot PC, click Run. Observe the robot start to run to ensure that there are no problems.

After the robot adds the 0.1N NaOH to the DNA in the AMP2 plate, the

Make AMP2 Wait for reaction time message appears. The wait time for this reaction is 10 minutes.

The robot PC sounds an alert and displays a message when the process is complete.

14. (Infinium LIMS only) Make sure the Use Barcodes check box is checked and click Run. a. Log in when prompted.

After the robot initializes, the Make AMP2 screen appears after a moment.

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Figure 85 Selecting Project or Batch for Make AMP2

b. Do one of the following:

— Select your current project. The available batches appear in the Sample

Batch ID pane. Select a batch to see the associated DNA plate appear in the DNA Plate pane.

Figure 86 Make AMP2 Screen with Project and Batch Selected

—

Use the Search box to search for a specific Batch ID or DNA Plate. c.

Select the batches you want to run and click OK.

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

15. Click OK in the message box. Remove the AMP2 plate from the robot bed and seal with the 96-well cap mat.

16. Invert the sealed AMP2 plate at least 10 times to mix the contents.

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Make the AMP2 Plate 93

17. Pulse centrifuge to 280 xg.

18. Record each DNA sample in the lab tracking worksheet.

19. Discard unused reagents in accordance with facility standards.

20. Proceed immediately to Incubate the AMP2 Plate on page 99.

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Make Multiple AMP2 Plates

This process creates up to four AMP2 plates for DNA amplification, allowing you to run 24, 48, 72, or 96 samples. First, DNA samples are added to designated well locations, and then 0.1N NaOH is added, denaturing the

DNA. Neutralization occurs when the MP1 reagent is added. AMM

(Amplification Master Mix) is then added to the DNA samples.

This feature may not be available on systems that are not running Infinium

LIMS (Laboratory Information Management System). For information about how to use Infinium LIMS, see the Infinium II LIMS User Guide.

Estimated Time

Robot:

`

35 minutes for 24 samples

`

70 minutes for 48 samples

`

105 minutes for 72 samples

`

140 minutes for 96 samples

Consumables

Item

MP1

AMM

0.1N NaOH

Quantity

3 tubes per

24 samples

3 tubes per

24 samples

15 ml per

24 samples

WG#-DNA plate, thawed to room temperature, with 24–

96 DNA samples

1 plate

96-well 0.8 ml microtiter plate

(MIDI)

1 plate for up to

24 samples; up to

4 plates total

Storage

-20°C

-20°C

4°C

22ºC

Supplied By

Illumina

Illumina

User

User

User

NOTE

Thaw all reagents completely at room temperature (22°C) and allow to equilibrate. Once thawed, gently invert each tube several times to thoroughly mix the reagent. Pulse centrifuge each tube to 280 xg to eliminate bubbles and collect reagent at the bottom of the tube.

Preparation

`

In preparation for the Incubate AMP2 process, preheat the Illumina

Hybridization Oven in the post-amp area to 37°C and allow the temperature to equilibrate.

`

Apply an AMP2 barcode label to each new storage plate.

`

In the Sample Sheet, enter the Sample_Name (optional) and

Sample_Plate for each Sample_Well.

Part # 11280749 Rev. A

Make Multiple AMP2 Plates 95

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• WG#-DNA plate barcode

• AMP2 plate barcodes

• MP1 tube barcode(s)

• AMM tube barcode(s)

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Prepare the Robot

For instructions on preparing the robot for use in a protocol, see the Infinium

II Assay Lab Setup and Procedures Guide.

Refer to Figure 87 throughout this protocol. Note that all of the barcodes

face to the right.

MP1 Tubes

AMM Tubes

NaOH in Quarter Reservoir

AMP2 Plates

WG#-DNA Plate Position

Figure 87 Tecan Eight-Tip Robot (Make Multi AMP2 Setup)

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Steps

1. If you do not already have a WG#-DNA plate, dispense DNA into either a:

• MIDI plate: 40 μl to each WG#-DNA plate well

• TCY plate: 30 μl to each WG#-DNA plate well

Apply a barcode label to the new WG#-DNA plate.

2. At the robot PC, select AMP2 Sample Prep Tasks | Make Multi AMP2.

3. In the DNA Plate Selection dialog box, select the plate type.

Do not mix plate types on the robot bed.

NOTE

4. If you are not using Infinium LIMS, skip ahead to step 6.

5. If you are using Infinium LIMS, click Run. a. Log in when prompted.

The Make Multi AMP2 screen appears after a moment.

Figure 88 Selecting Project for Make Multi AMP2

b. Scan or type the DNA plate barcode into the Search For text box, and then click Search.

c.

Select up to four batches and click OK.

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Make Multiple AMP2 Plates 97

Figure 89 Selecting Batches

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

6. (Non-Infinium LIMS only) In the Basic Run Parameters pane, enter the

Number of DNA samples (24, 48, 72, or 96).

NOTE

If you are using Infinium LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

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Figure 90 Make Multi AMP2 Screen

7. Vortex the sealed WG#-DNA plate at 1600 rpm for 1 minute.

8. Centrifuge to 280 xg for 1 minute at 22°C.

9. Remove the cap mat.

10. Place the MP1 and AMM tubes in the robot tube rack according to the

bed map (Figure 90). Remove the caps.

11. Add 15 ml NaOH to the quarter reservoir, and then place the reservoir on

the robot bed according to the bed map (Figure 90).

12. Place the AMP2 and WG#-DNA plates on the robot bed according to the

bed map (Figure 90).

Infinium LIMS only: The robot starts the process when the plates are in place.

13. In the lab tracking worksheet, record the plate positions on the robot bed.

14. Make sure that all items are placed properly on the robot bed, that all caps and seals have been removed, and that all the barcodes face to the right.

15. (Non-Infinium LIMS only) At the robot PC, clear the Use Barcodes check box and click Run. Observe the robot start to run to ensure that there are no problems.

After the robot has added the 0.1N NaOH to the DNA in the AMP2 plates, the Make Multi AMP2 Wait for reaction time message appears.

Wait time for this reaction is 10 minutes per plate.

The robot PC sounds an alert and displays a message when the process is complete.

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Incubate the AMP2 Plate 99

16. Click OK in the message box. Remove the AMP2 plates from the robot bed and seal them with the 96-well cap mats.

17. Invert each sealed AMP2 plate at least 10 times to mix contents.

18. Pulse centrifuge the plates to 280 xg.

19. Discard unused reagents in accordance with facility standards.

20. Proceed immediately to Incubate the AMP2 Plate on page 99.

Incubate the AMP2 Plate

This process incubates the AMP2 plate for 20–24 hours at 37°C in the

Illumina Hybridization Oven. It generates a sufficient quantity of each individual DNA sample to be used twice in the Two-Sample HC Infinium II

Assay.

Figure 91 Incubating DNA to Amplify

Estimated Time

20–24 hours.

Preparation Verify AMP2 for Incubation (Infinium LIMS only)

1. In the Infinium LIMS left sidebar, click Infinium II HC Multi-Sample |

Incubate AMP2.

2. Scan the barcode of the AMP2 plate and click Verify.

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Figure 92 (Infinium LIMS) Verifying AMP2 for Incubation

3. If the AMP2 plate is queued for incubation, a blue confirmation message appears at the top of the window. Proceed with incubation.

4. If the AMP2 plate is not queued for incubation, a red error message appears at the top of the window. Do

not

proceed with incubation.

Instead, follow these steps to troubleshoot the problem: a. Click the Reports tab in the upper-right corner.

b. In the left sidebar, click Tracking Reports | Get Queue Status. c.

Scan the plate barcode and click Go. d. Note what step the plate is queued for, and proceed with that step.

For information about how to use Infinium LIMS, see the Infinium II LIMS User

Guide.

Steps

1. Incubate the AMP2 plate in the Illumina Hybridization Oven for at least

20 but no more than 24 hours at 37°C.

2. On the lab tracking worksheet, record the start and stop times.

3. If you are using Infinium LIMS: a. In the Infinium LIMS left sidebar, click Infinium II HC Multi-Sample |

Incubate AMP2. b. Scan the barcode of the AMP2 plate and click Save. Infinium LIMS records the data and queues the plate for fragmentation.

4. Proceed to Fragment the AMP2 Plate on page 101.

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Fragment the AMP2 Plate 101

Fragment the AMP2 Plate

This process enzymatically fragments the amplified DNA samples. An endpoint fragmentation is used to prevent over-fragmentation.

Figure 93 Fragmenting DNA

Estimated Time

Robot time:

• 10 minutes for 8 samples

• 50 minutes for 48 samples

• 100 minutes for 96 samples

Consumables

Item

FRG

Quantity Storage

1 tube per 8 samples -20°C

Supplied By

Illumina

NOTE

Thaw all reagents completely at room temperature (22°C) and allow to equilibrate. Once thawed, gently invert each tube several times to thoroughly mix the reagent. Pulse centrifuge each tube to 280 xg to eliminate bubbles and collect reagent at the bottom of the tube.

Preparation

`

Preheat the heat block with the MIDI plate insert to 37°C.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• FRG tube barcode(s)

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

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Prepare the

Robot

For instructions on preparing the robot for use in a protocol, see the Infinium

II Assay Lab Setup and Procedures Guide.

Refer to Figure 94 throughout this protocol. Note that the barcodes all face

to the right.

FRG Tube

AMP2 Plate

Figure 94 Tecan Eight-Tip Robot (Fragment AMP2 Setup)

Steps Set Up the Robot

1. Centrifuge the AMP2 plate to 50 xg for 1 minute.

2. At the robot PC, select AMP2 Sample Prep Tasks | Fragment AMP2.

3. (Non-Infinium LIMS only) In the Basic Run Parameters pane, enter the

Number of DNA samples and the Number of AMP2 Plates (1 to 4).

NOTE

If you are using Infinium LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

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Fragment the AMP2 Plate 103

Figure 95 Fragment AMP2 Screen

4. Place the AMP2 plate on the robot bed according to the robot bed map

(Figure 95). Remove any plate seals.

5. Place the FRG tubes in the robot tube rack according to the robot bed map. Remove the caps.

6. In the lab tracking worksheet, record the plate positions on the robot bed.

7. Make sure that all items are placed properly on the robot bed, that all caps and seals have been removed, and that all the barcodes face to the right.

Start the Robot

1. At the robot PC: a. If you are not running Infinium LIMS, clear the Use Barcodes check box. b. Click Run to start the process. c.

Log in if prompted.

d. Observe the robot start to run to ensure that there are no problems.

The robot PC sounds an alert and displays a message when the process is complete.

2. Click OK in the message box. Remove the AMP2 plate from the robot bed and seal with the 96-well cap mat.

3. Vortex the plate at 1600 rpm for 1 minute.

4. Centrifuge to 50 xg for 1 minute at 22°C.

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5. Incubate on the heat block for 1 hour at 37°C.

6. On the lab tracking worksheet, record the start and stop times.

7. Discard unused reagents in accordance with facility standards.

8. Do one of the following:

•

Proceed to Precipitate the AMP2 Plate on page 104. Leave plate in

the 37°C heat block until preparation is complete.

• Store the sealed AMP2 plate at -20°C if you do not plan to proceed to the next step immediately.

This is a good stopping point in the process.

Precipitate the AMP2 Plate

In this process, PA1 and 2-propanol are added to the AMP2 plate to precipitate the DNA samples.

Figure 96 Precipitating DNA

Estimated Time

Robot time:

• 20 minutes for 8 samples

• 70 minutes for 48 samples

• 140 minutes for 96 samples

Consumables

Item

PA1

100% 2-propanol

Quantity Storage

1 tube per 8 samples 4°C

1 bottle Room temperature

Supplied By

Illumina

User

Preparation

`

If you froze the AMP2 plate after fragmentation, thaw it to room temperature (22°C). Centrifuge to 280 xg for 1 minute.

`

Preheat the heat block to 37°C, if it is not already.

Part # 11280749 Rev. A

Precipitate the AMP2 Plate 105

`

In preparation for the 4°C spin, set the centrifuge to 4°C.

`

Thaw PA1 to room temperature (22°C). Centrifuge to 280 xg for

1 minute.

`

Preheat the heat sealer. Allow 20 minutes.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• PA1 tube barcode(s)

• 2-propanol lot number and date opened

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Prepare the

Robot

For instructions on preparing the robot for use in a protocol, see the Infinium

II Assay Lab Setup and Procedures Guide.

Refer to Figure 97 throughout this protocol. Note that all of the barcodes

face to the right.

2-propanol in Full Reservoir

PA1 in Half Reservoir

AMP2 Plate

Figure 97 Tecan Eight-Tip Robot (Precip AMP2 Setup)

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Verify AMP2 for Centrifugation (Infinium LIMS only)

1. In the Infinium LIMS left sidebar, click Infinium II HC Multi-Sample |

Spin AMP2.

2. Scan the barcode of the AMP2 plate and click Verify. You can scan up to four plates.

Figure 98 (Infinium LIMS) Verifying AMP2 for Centrifugation

3. If the AMP2 plate is queued for centrifugation, a blue confirmation message appears at the top of the window. Proceed to precipitation.

4. If the AMP2 plate is not queued for centrifugation, a red error message appears at the top of the window. Do not proceed with centrifugation.

Instead, follow these steps to troubleshoot the problem: a. Click the Reports tab in the upper-right corner.

b. In the left sidebar, click Tracking Reports | Get Queue Status. c.

Scan the plate barcode and click Go. d. Note what step the plate is queued for, and proceed with that step.

For information about how to use Infinium LIMS, see the Infinium II LIMS User

Guide.

Steps Set Up the Robot

1. At the robot PC, select AMP2 Sample Prep Tasks | Precip AMP2.

2. (Non-Infinium LIMS only) In the Basic Run Parameters pane, enter the

Number of DNA samples and the Number of AMP2 plates (1 to 4).

Part # 11280749 Rev. A

Precipitate the AMP2 Plate 107

NOTE

If you are using Infinium LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

Figure 99 Precip AMP2 Screen

3. Centrifuge the sealed AMP2 plate to 50 xg for 1 minute at 22°C.

4. Place the AMP2 plate on the robot bed according to the robot bed map

(Figure 99). Remove the plate seal.

5. Place a half reservoir in the reservoir frame, according to the robot bed

map (Figure 99), and add PA1 as follows:

• 8 samples: 1 tube

• 48 samples: 6 tubes

• 96 samples: 12 tubes

6. Place a full reservoir in the reservoir frame, according to the robot bed map, and add 2-propanol as follows:

• 8 samples: 12 ml

• 48 samples: 74 ml

• 96 samples: 142 ml

7. In the lab tracking worksheet, record the plate position on the robot bed.

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8. Make sure that all items are placed properly on the robot bed, that all caps and seals have been removed, and that all the barcodes face to the right.

Start the Robot

1. At the robot PC: a. If you are not running Infinium LIMS, clear the Use Barcodes check box. b. Click Run to start the process. c.

Log in if prompted.

d. Observe the robot start to run to ensure that there are no problems.

2. When prompted, remove the AMP2 plate from the robot bed. Do

not

click OK in the message box yet.

3. Seal the AMP2 plate with the same cap mat removed earlier.

4. Vortex the plate at 1600 rpm for 1 minute.

5. Incubate at 37°C for 5 minutes.

6. Centrifuge to 50 xg at room temperature (22°C) for 1 minute.

7. Return the AMP2 plate to the robot bed according to the robot bed map

(Figure 99). Remove the plate seal.

8. At the robot PC, click OK to restart the run.

The robot PC sounds an alert and displays a message when the process is complete.

9. Click OK in the message box. Remove the AMP2 plate from the robot bed and carefully seal with a

new, dry

cap mat, taking care not to shake the plate in any way until the cap mat is fully seated.

10. Invert each plate at least 10 times to mix contents thoroughly.

11. Incubate for 30 minutes at 4°C.

12. Place the sealed AMP2 plate in the centrifuge opposite another plate of

equal weight (Figure 100).

Part # 11280749 Rev. A

Precipitate the AMP2 Plate 109

Plate of Equal Weight for Balance

Sealed AMP2 Plate

Figure 100 Balancing AMP2 Plate in Centrifuge

13. Centrifuge to 3000 xg for 20 minutes at 4°C.

14. Immediately remove the AMP2 plate from the centrifuge.

Perform the next step immediately to avoid dislodging the blue pellet. If any delay occurs, repeat steps 13 through 14 before proceeding.

15. Remove the cap mat and discard it.

16. Decant supernatant by quickly inverting the AMP2 plate and smacking it down onto an absorbent pad appropriate for 2-propanol disposal.

CAUTION

To ensure optimal performance, do not allow supernatant in wells to pour into other wells. Keep the plate inverted.

17. Tap the plate firmly on the pad several times over a period of 1 minute or until all wells are completely devoid of liquid.

18. Place the inverted, uncovered plate on a tube rack for 1 hour at 22°C to

air dry the pellet (Figure 101).

At this point, blue pellets should be present at the bottoms of the wells.

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Figure 101 Uncovered AMP2 Plate Inverted for Air Drying

CAUTION

Do not over-dry the pellet. Pellets that are over-dried will be difficult to resuspend. Poorly resuspended samples will lead to poor genotyping results.

19. On the lab tracking worksheet, record the start and stop times.

20. If you are using Infinium LIMS: a. In the Infinium LIMS left sidebar, click Infinium II HC Multi-Sample |

Spin AMP2. b. Scan the barcode of the AMP2 plate and click Save. Infinium LIMS records the data and queues the plate for resuspension.

21. Discard unused reagents in accordance with facility standards.

22. Do one of the following:

•

Proceed immediately to Resuspend the AMP2 Plate on page 111.

• Heat-seal the AMP2 plate and store it at -20°C for the following day or -80°C for long-term storage.

This is a good stopping point in the process.

Part # 11280749 Rev. A

Resuspend the AMP2 Plate 111

Resuspend the AMP2 Plate

In this process, RA1 is added to the AMP2 plate to resuspend the precipitated DNA samples.

Figure 102 Resuspending DNA

Estimated Time

Robot time: 5 minutes per plate

Incubation time: 1 hour

Consumables

Item

RA1

Quantity

Bottle (4 ml)

Storage

-20°C

Supplied By

Illumina

WARNING

This protocol involves the use of an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. Refer to the Infinium Assay

MSDS on your Documentation CD for complete information.

Preparation

`

Gradually warm the RA1 reagent to room temperature, preferably in a

20–25°C water bath. Gently mix to dissolve any crystals that may be present.

`

If you stored the AMP2 plate at -20°C, thaw it to room temperature.

Remove the cap mat and discard it.

`

Preheat the Illumina Hybridization Oven to 48°C.

`

Preheat the heat sealer. Allow 20 minutes.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• RA1 bottle barcode(s)

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NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Prepare the

Robot

For instructions on preparing the robot for use in a protocol, see the Infinium

II Assay Lab Setup and Procedures Guide.

Refer to Figure 103 throughout this protocol. Note that all of the barcodes

face to the right.

RA1 in Quarter

Reservoir

AMP2 Plate

Figure 103 Tecan Eight-Tip Robot (Resuspend AMP2 Setup)

Steps Set Up the Robot

1. At the robot PC, select AMP2 Sample Prep Tasks | Resuspend AMP2.

2. (Non-Infinium LIMS only) In the Basic Run Parameters pane, enter the

Number of DNA samples and the Number of AMP2 plates (1 to 4).

NOTE

If you are using Infinium LIMS, you cannot change the number of DNA samples on this screen. However, the LIMS software processes the correct number of samples.

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

Part # 11280749 Rev. A

Resuspend the AMP2 Plate 113

Figure 104 Resuspend AMP2 Screen

3. Place the AMP2 plate on the robot bed according to the robot bed map

(Figure 104). Remove the plate seal.

4. Place a quarter reservoir in the reservoir frame, according to the robot bed map, and add RA1 as follows:

• 4 ml for 8 samples

• 8 ml for 16 samples

• 12 ml for 24 samples

5. In the lab tracking worksheet, record the plate positions on the robot bed.

6. Make sure that all items are placed properly on the robot bed, that all caps and seals have been removed, and that all the barcodes face to the right.

Start the Robot

1. At the robot PC: a. If you are not running Infinium LIMS, clear the Use Barcodes check box. b. Click Run to start the process. c.

Log in if prompted.

d. Observe the robot start to run to ensure that there are no problems.

The robot PC sounds an alert and displays a message when the process is complete.

2. When prompted, remove the AMP2 plate from the robot bed and click

OK.

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3. Apply a foil heat seal to the AMP2 plate by firmly holding the heat sealer sealing block down for 3 seconds.

4. Place the sealed plate in the Illumina Hybridization Oven and incubate for 1 hour at 48°C.

5. In the lab tracking worksheet, record the start and stop times.

6. Vortex the sealed plate at 1800 rpm for 1 minute.

7. Pulse centrifuge to 280 xg.

CAUTION

If you stored the DNA pellets at -20°C for more than

72 hours after Precip AMP2, you may need to repeat the vortexing and centrifugation until the pellets are completely resuspended.

8. Discard unused reagents in accordance with facility standards.

9. Do one of the following:

•

Proceed to Hybridize HC BC2 on page 115. If you do so

immediately, it is safe to leave the RA1 at room temperature.

• Store the sealed AMP2 plate and the RA1 at -20°C (-80°C if storing for more than 24 hours).

This is a good stopping point in the process.

Part # 11280749 Rev. A

Hybridize HC BC2 115

Hybridize HC BC2

In this process, the resuspended DNA samples are denatured and dispensed onto BeadChips using the robot. DNA-loaded BeadChips are placed into

Hyb Chamber Inserts, which are then positioned into the Hyb Chambers.

The Hyb Chambers are hybridized in the Illumina Hybridization Oven for 16–

24 hours at 48°C.

(g DN A )

(id ent ical p r o b es p er b ead t y p e)

(g DN A )

Figure 105 Hybridizing DNA to BeadChip

Estimated Time

Robot time:

• 8 samples: 10 minutes

• 48 samples: 30 minutes

• 96 samples: 60 minutes

Incubation time: 16–24 hours

Consumables

Item

PB2

Quantity

(per 8 Samples)

1 tube

BeadChips

Hyb Chambers

Hyb Chamber gaskets

Hyb Chamber inserts

Robot BeadChip Alignment

Fixture

1

4

4

1

2

Storage Supplied By

Room temperature

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

NOTE

Thaw all reagents completely at room temperature (22°C) and allow to equilibrate. Once thawed, gently invert each tube several times to thoroughly mix the reagent. Pulse centrifuge each tube to 280 xg to eliminate bubbles and collect reagent at the bottom of the tube.

Preparation `

Preheat the heat block to 95°C.

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`

Preheat the Illumina Hybridization Oven to 48°C and set the rocker speed to 5.

`

If you plan to perform the XStain process tomorrow, begin thawing the

XC4 reagent. For instructions, see Resuspend XC4 Reagent for XStain

BC2 on page 128.

`

Prepare the Illumina Hybridization Oven as follows: a. Preheat the oven to 48°C:

—

Press the "F" button once to change the display to TSET.

— Press the "S" button to enter the set-temperature mode, and then use the Increment/Decrement dial to set the oven to 48°C.

— Press the "S" button again to set 48°C as the temperature.

b. Set the rocker speed to 5:

—

Press the "F" button twice until SPd is indicated on the display.

— Press the "S" button to enter the rocker speed mode.

— Use the Increment/Decrement dial to set the rocker speed to "5".

— Press the "S" button again.

`

Calibrate the Illumina Hybridization Oven with the Full-Scale Plus digital thermometer supplied with your system.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• PB2 tube barcode(s)

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Prepare Hyb Chambers

NOTE

Perform the Hyb Chamber assembly near the Hyb Oven to minimize the distance you need to move the BeadChiploaded Hyb Chamber. Take care to keep the Hyb Chamber steady and level when lifting and moving. Avoid shaking and keep the Hyb Chamber parallel to the lab bench at all times.

1. For every 4 BeadChips, place the following items on the bench top

(Figure 106):

• BeadChip Hyb Chambers (1)

• Hyb Chamber Gaskets (1)

• Robot BeadChip Alignment Fixtures (2)

• BeadChip Hyb Chamber Inserts (4)

Part # 11280749 Rev. A

Hyb Chamber

Hybridize HC BC2 117

Robot BeadChip

Alignment Fixture

Hyb Chamber

Gasket

Hyb Chamber

Inserts

Figure 106 BeadChip Hyb Cartridge Components

Barcode Ridges

2. Place the BeadChip Hyb Chamber gasket into the Hyb Chambers: a. Match the wider edge of the Hyb Chamber gasket to the barcode-

ridge side of the Hyb Chamber (Figure 107).

Wider Edge

Reservoirs Narrower Edge

Figure 107 Hyb Chamber and Gasket

b. Lay the gasket into the Hyb Chamber (Figure 108), and then press it

down all around.

Figure 108 Placing Gasket into Hyb Chamber

Infinium II Assay Two-Sample HC Protocols

118 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol c.

Make sure the Hyb Chamber gasket is properly seated (Figure 109).

Figure 109 Hyb Chamber with Gasket in Place

3. Dispense 200 μl PB2 into each of the 8 humidifying buffer reservoirs in

the Hyb Chamber (Figure 110).

Figure 110 Dispensing PB2 into Hyb Chamber Reservoirs

4. Remove the BeadChips from 4°C storage but do not unpackage.

5. Close and lock the BeadChip Hyb Chamber lid (Figure 111).

a. Seat the lid securely on the bottom plate.

b. Snap two clamps shut, diagonally across from each other.

c.

Snap the other two clamps.

Part # 11280749 Rev. A

Hybridize HC BC2 119

Figure 111 Sealing the Hyb Chamber

6. Leave the closed Hyb Chamber on the bench at room temperature until the BeadChips are loaded with DNA sample.

Prepare the Robot

For instructions on preparing the robot for use in a protocol, and ensuring that the Chamber Rack is properly installed on the post-amplification robot bed, see the Infinium II Assay Lab Setup and Procedures Guide.

Infinium II Assay Two-Sample HC Protocols

120 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Refer to Figure 112 throughout this protocol. Note that all of the plate

barcodes face to the right.

AMP2 Plate

Robot BeadChip

Alignment Fixtures

Figure 112 Placing Alignment Fixtures and AMP2 Plate onto Robot Bed

Verify AMP2 and BeadChips for Hyb (Infinium

LIMS only)

1. In the Infinium LIMS left sidebar, click Infinium II HC Multi-Sample |

Confirm BeadChips for Hyb.

2. Scan the barcode of the AMP2 plate.

3. Scan the barcodes of all the BeadChips you plan to hybridize with the plate. You can scan up to 24 BeadChips.

NOTE

Only scan BeadChips that have been accessioned into the system. The BeadChip type must match the type associated with this batch in Infinium LIMS.

4. Click Verify.

Part # 11280749 Rev. A

Hybridize HC BC2 121

Figure 113 (Infinium LIMS) Verifying AMP2 and BeadChips for Hyb

5. If the AMP2 plate and BeadChips are queued for hybridization, a blue confirmation message appears at the top of the window. Proceed to loading the BeadChips.

If the AMP2 plate is not queued for hybridization, if any of the BeadChips have not been accessioned into the system, or if any of the BeadChips are the wrong type, a red error message appears at the top of the window. The error message indicates the first incorrect barcode it finds. Do

not

proceed with hybridization.

6. If the plate is not queued for hybridization: a. Click the Reports tab in the upper-right corner.

b. In the left sidebar, click Tracking Reports | Get Queue Status. c.

Scan the plate barcode and click Go. d. If the plate is queued for another step, proceed with that step.

7. If one of the BeadChips is not accessioned into the system, accession it and then repeat the verification step.

8. If one of the BeadChips is not the right type for this batch, accession one that is the right type and repeat the verification step.

9. When the verification is successful, proceed to Steps.

For information about how to use Infinium LIMS, see the Infinium II LIMS User

Guide.

Infinium II Assay Two-Sample HC Protocols

122 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Steps Load the BeadChips

1. Place the resuspended AMP2 plate on the heat block at 95°C for

20 minutes, to denature the samples.

2. Vortex the AMP2 plate at 1800 rpm.

3. Pulse centrifuge the AMP2 plate to 280 xg for 1 minute.

CAUTION

Do not unpackage the BeadChips until you are ready to begin hybridization.

4. Remove all BeadChips from their packages.

5. Slide each BeadChip into the Robot BeadChip Alignment Fixture(s) so that the barcode lines up with the ridges on the fixture. You need

4 BeadChips for 8 samples and 12 BeadChips for 24 samples.

CAUTION

Handle BeadChips only by the edges or by the barcode end.

Figure 114 Placing BeadChips into Robot Alignment Fixture

6. Stack the Robot BeadChip Alignment Fixtures and carry them to the robot.

Part # 11280749 Rev. A

Hybridize HC BC2 123

Figure 115 Four Stacked Robot Alignment Fixtures

Set Up the Robot

1. At the robot PC, select AMP2 Hyb Tasks | Hyb HC BC2.

2. (Non-LIMS only) In the Basic Run Parameters pane, enter the Number of

DNA samples.

You can only dispense samples from one AMP2 plate at a time. To pro-

cess additional AMP2 plates, repeat the Steps.

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

Figure 116 Hyb HC BC2 Screen

3. Place the Robot BeadChip Alignment Fixtures onto the robot bed

according to the bed map (Figure 116). Do one of the following:

• For 8 samples, position the two Robot BeadChip alignment fixtures in the top row.

Infinium II Assay Two-Sample HC Protocols

124 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

• For 24 samples, use all six positions on the robot bed.

4. Place the AMP2 plate onto the robot bed according to the bed map and remove the foil seal.

5. On the lab tracking worksheet, record the plate and fixture positions on the robot bed, and record the BeadChip barcodes associated with each well.

6. Make sure that all items are placed properly on the robot bed, that all caps and seals have been removed, and that all the barcodes face to the right.

Start the Robot

The robot PC sounds an alert and displays a message when the process is complete.

7. When prompted, click OK.

8. Remove the Robot BeadChip Alignment Fixtures from the robot bed and visually inspect all sections of the BeadChips. Record any beadstripe sections that are not completely covered by DNA sample.

9. Apply a foil heat-seal to the AMP2 plate by firmly holding the heat sealer block down for 3 seconds.

10. Store the AMP2 plate at -20°C, or at -80 °C if you do not plan to use it again within 24 hours.

11. Continue on to Set Up HC BC2 for Hyb on page 124.

Set Up HC BC2 for Hyb

1. Ensure the Illumina Hybridization Oven is set to 48°C.

CAUTION

Hold the BeadChip by the ends with your thumb and forefinger (thumb at the barcode end). Do not hold the

BeadChip by the sides near the sample inlets. Avoid contacting the beadstripe area and sample inlets.

2. Carefully remove each BeadChip from the Robot BeadChip Alignment

Fixtures when the robot finishes.

3. Carefully place each BeadChip in a Hyb Chamber insert, orienting the

barcode end so that it matches the barcode symbol on the insert (Figure

117).

CAUTION

For optimal performance, take care to keep the Hyb

Chamber inserts containing BeadChips steady and level when lifting or moving. Avoid shaking and keep parallel to the lab bench at all times. Do not hold by the sides near the sample inlets.

Part # 11280749 Rev. A

Hybridize HC BC2 125

Figure 117 Placing BeadChips into Hyb Chamber Inserts

4. Load the Hyb Chamber inserts containing BeadChips into the Hyb

Chambers (Figure 118). Position the barcode end over the ridges

indicated on the Hyb Chamber.

Figure 118 Placing Hyb Chamber Inserts into Hyb Chamber

5. Place the back side of the lid onto the Hyb Chamber and then slowly bring down the front end to avoid dislodging the Hyb Chamber inserts

(Figure 119).

6. Close the clamps on both sides of the Hyb Chamber.

Figure 119 Securing Hyb Chamber Lid

Infinium II Assay Two-Sample HC Protocols

126 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

NOTE

For optimal performance, take care to keep the Hyb

Chamber steady and level when lifting or moving. Avoid shaking the Hyb Chamber, and keep the Hyb Chamber parallel to the lab bench while you transfer it to the

Illumina Hybridization Oven.

7. Place the Hyb Chamber in the 48°C Illumina Hybridization Oven so that the clamps of the Hyb Chamber face the left and right side of the oven.

The Illumina logo on top of the Hyb Chamber should be facing you.

NOTE

If you are stacking multiple Hyb Chambers in the Illumina

Hybridization Oven, make sure the feet of the top Hyb

Chamber fit into the matching indents on top of the bottom Hyb Chamber. This will hold the Hyb Chambers in place while they are rocking.

Figure 120 Hyb Chamber Correctly Placed in Hyb Oven

Part # 11280749 Rev. A

Hybridize HC BC2 127

Figure 121 Two Hyb Chambers Correctly Placed in Hyb Oven

Figure 122 Incorrectly Placed Hyb Chamber

8. (Optional) Set the rocker speed to 5 and start the rocker.

9. Incubate the Hyb Chamber(s) in the Illumina Hybridization Oven for 16–

24 hours at 48°C.

10. On the lab tracking worksheet

,

enter the start and stop times.

Infinium II Assay Two-Sample HC Protocols

128 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

11. Place RA1 into the freezer at -20°C for use the next day.

Resuspend XC4 Reagent for XStain BC2

Keep the XC4 in the bottle in which it was shipped until ready for use. In preparation for the XStain protocol, follow these steps to resuspend the XC4 reagent:

1. Add 330 ml 100% EtOH to the XC4 bottle. The final volume will be

350 ml.

Each XC4 bottle (350 ml) has enough solution to process up to 24 Bead-

Chips.

2. Shake vigorously for 15 seconds.

3. Leave the bottle upright on the lab bench overnight.

NOTE

If the XC4 was not left to resuspend overnight, you can still proceed with the assay. Add the EtOH and put the XC4 on its side on a rocker to resuspend. Leave it there until the

BeadChips are ready for coating.

4. Shake again to ensure that the pellet is completely resuspended. If any coating is visible, vortex at 1625 rpm until it is in complete suspension.

Once resuspended with 330 ml 100% EtOH, use XC4 at room temperature. You can store it at 4°C overnight, but thaw it again before use.

5. Proceed to Wash BC2 on page 129.

Part # 11280749 Rev. A

Wash BC2 129

Wash BC2

In this process, the BeadChips are prepared for the XStain BC2 process. The coverseals are removed from the BeadChips, and the BeadChips are washed in WB1 reagent followed by PB1 reagent to remove unhybridized and nonspecifically hybridized DNA. The BeadChips are then assembled into Flow-

Through Chambers under the PB1 buffer.

Figure 123 Washing BeadChip

Estimated Time

Robot time:

• 4 BeadChips: 20 minutes

• 24 BeadChips: 50 minutes

Consumables

Item

Quantity

(per 8 Samples)

Storage

PB1

WB1

Wash Rack

350 ml per

8 BeadChips

1 bottle per 8

BeadChips

1 Multi-Sample BeadChip

Alignment Fixture

Te-Flow Flow-Through

Chambers (with Black Frames,

Spacers, Glass Back Plates, and Clamps)

1 per BeadChip (4 or

24)

Wash Dish 4 BeadChips: 1 dish

24 BeadChips: 3 dishes

4 BeadChips: 1 rack

24 BeadChips: 3 racks

Room temperature

-20°C

Supplied By

Illumina

Illumina

Illumina

Illumina

Illumina

Illumina

Infinium II Assay Two-Sample HC Protocols

130 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

WARNING

This protocol involves the use of an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. Refer to the Infinium Assay

MSDS on your Documentation CD for complete information.

NOTE

Thaw all reagents completely at room temperature (22°C) and allow to equilibrate. Once thawed, gently invert each tube several times to thoroughly mix the reagent. Pulse centrifuge each tube to 280 xg to eliminate bubbles and collect reagent at the bottom of the tube.

Preparation

`

Thaw WB1 to room temperature (22°C) and allow to equilibrate. Ensure that the solution is completely redissolved.

`

Fill 1–3 wash dishes with 200 ml WB1. Label each dish.

`

Fill 1–3 wash dishes with 200 ml PB1. Label each dish.

`

Fill the BeadChip Alignment Fixture with 150 ml PB1.

`

Lay out the Te-Flow Flow-Through Chamber components (black frames, spacers, clean glass back plates, and clamps) on the benchSeparate the clear plastic spacers from the white backs.

`

Clean the glass back plates as directed in the Infinium II Assay Lab Setup

and Procedures Guide.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• WB1 bottle barcode

• PB1 bottle barcode

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Verify Reagents and BeadChips for Washing (Infinium

LIMS only)

1. In the Infinium LIMS left sidebar, click Infinium II HC Multi-Sample |

Wash BC2.

2. Scan the barcode(s) of the PB1.

3. Scan the barcode(s) of the WB1.

Part # 11280749 Rev. A

4. Scan the BeadChip barcodes.

5. Click Verify.

Wash BC2 131

Figure 124 (Infinium LIMS) Verifying Reagents and BeadChips for Washing

6. If the reagents are correct and BeadChips are queued for washing, a blue confirmation message appears at the top of the window. Proceed to the wash step.

7. If any of the reagents are invalid, check the reagent type before rescanning. The reagent name (e.g., PB1) appears at the end of the barcode. Make sure to scan the correct reagent into each box.

8. If any of the BeadChips are not queued for washing, a red error message appears at the top of the window. The error message indicates the first incorrect barcode it finds. Do not proceed with washing. Instead, follow these steps to troubleshoot the problem: a. Click the Reports tab in the upper-right corner.

b. In the left sidebar, click Tracking Reports | Get Queue Status. c.

Scan the BeadChip barcode that appeared in the error message and click Go. d. Note what step the BeadChip is queued for, and proceed with that step.

For information about how to use Infinium LIMS, see the Infinium II LIMS User

Guide.

Infinium II Assay Two-Sample HC Protocols

132 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Steps Wash BC2

1. Attach the wire handle to the wash rack and submerge it in the WB1

wash dish (Figure 125).

Figure 125 Wash Rack in WB1 Wash Dish

2. For each Hyb Chamber insert: a. Remove it from the Hyb Chamber. b. Remove the BeadChip from the insert.

Do not touch the arrays!

CAUTION c.

Using powder-free gloves and pulling from the barcode end, remove the coverseal from the BeadChip by stripping it off in a downward direction.

CAUTION

To ensure no solution splatters on you, Illumina recommends removing the coverseal over an absorbent cloth or paper towels, preferably in a hood.

Figure 126 Removing the Coverseal

Part # 11280749 Rev. A

Wash BC2 133 d. Immediately and carefully slide the BeadChip into the wash rack, making sure that the BeadChip is completely submerged in the WB1

(Figure 127).

Figure 127 Placing BeadChips in WB1 Wash Dish

3. Repeat step 2 until all BeadChips (maximum of 8) are submerged in the wash rack.

4. Move the wash rack up and down for 1 minute, breaking the surface of the WB1 with gentle, slow agitation.

5. Move the wash rack to the wash dish containing PB1. Make sure that the

BeadChips are completely submerged.

6. Move the wash rack up and down for 1 minute, breaking the surface of the PB1 reagent with gentle, slow agitation.

7. If you are processing more than 8 BeadChips, repeat steps 2 through 6.

8. Immediately wash the Hyb Chamber reservoirs with dH

2

O and scrub them clean with a small brush, ensuring that no PB2 remains.

CAUTION

It is important to wash the reservoirs immediately and thoroughly to ensure that no traces of PB2 remain in the wells.

Infinium II Assay Two-Sample HC Protocols

134 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Assemble Flow-Through Chamber

1. For each BeadChip, place a black frame into the BeadChip Alignment

Fixture (Figure 128). The fixture should be pre-filled with 150 ml PB1.

Figure 128 Placing Black Frames into Alignment Fixture

2. Place each BeadChip into a black frame. Align its barcode with the ridges

stamped onto the Alignment Fixture (Figure 129).

Figure 129 Placing BeadChip into Black Frame on Alignment Fixture

3. Place a

clear

spacer onto the top of each BeadChip (Figure 130). Use the

Alignment Fixture grooves to guide the spacers into proper position.

Be sure to use the clear plastic spacers, not the white ones.

NOTE

Part # 11280749 Rev. A

Groove for

Positioning Spacer

White Spacer

Do Not Use

Clear Spacer

Wash BC2 135

Figure 130 Placing Clear Plastic Spacer onto BeadChip

4. Place the Alignment Bar onto the Alignment Fixture (Figure 131), fitting

the groove in the Alignment Bar over the metal tab on the Alignment

Fixture.

Figure 131 Placing Alignment Bar onto Alignment Fixture

5. Use a Whoosh duster or laboratory air gun to quickly remove any accumulated dust from the glass back plates just before placing them onto the BeadChips.

6. Place a clean glass back plate on top of the clear spacer covering each

BeadChip. The plate reservoir should be at the barcode end of the

Infinium II Assay Two-Sample HC Protocols

136 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

BeadChip, facing inward to create a reservoir against the BeadChip

surface (Figure 132).

Reservoir at Barcode End of Glass Back Plate

Glass Back Plate in Position

Figure 132 Placing Glass Back Plate onto BeadChip

7. Attach the metal clamps as follows (Figure 133):

a. Gently push the glass back plate up against the Alignment Bar with one finger.

b. Place the first metal clamp around the Flow-Through Chamber so that one stripe shows between it and the Alignment Bar.

c.

Place the second metal clamp around the Flow-Through Chamber at the barcode end, just below the reagent reservoir, so that no stripes show between the clamp and the barcode.

Part # 11280749 Rev. A

Wash BC2 137

One Stripe Shows

Between First Clamp and Alignment Bar

Glass Back Plate

Pressed Against

Alignment Bar

No Stripes Show

Between Second

Clamp and Barcode

Figure 133 Securing Flow-Through Chamber Assembly with Metal Clamps

8. With scissors, trim the spacer at the non-barcode end of the assembly

(Figure 134). Slip scissors up over the barcode to trim the other end.

Trim Spacer at Non-Barcode

End of Flow-Through Chamber

Trim Spacer at Barcode End of

Flow-Through Chamber

Figure 134 Trimming Spacer Ends from Flow-Through Chamber Assembly

9. Discard unused reagents in accordance with facility standards.

10. If you are using Infinium LIMS: a. In the Infinium LIMS left sidebar, click Infinium II HC Multi-Sample |

Wash BC2.

Infinium II Assay Two-Sample HC Protocols

138 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol b. Scan the reagent barcodes and BeadChip barcodes. Click Save.

Infinium LIMS records the data and queues the BeadChips for the

next step, Single-Base Extension & Stain BC2.

11. Proceed to Single-Base Extension & Stain BC2 on page 138.

Single-Base Extension & Stain BC2

(g DN A )

A

T*

C*

G

(g DN A )

* St ain in red channel

* St ain in green channel

Figure 135 Extending and Staining BeadChip

Estimated Time

Robot time:

• 2 hours and 10 minutes per 8 samples

• 2 hours and 40 minutes per 24 samples

Consumables

Following hybridization, RA1 reagent is used to wash away any remaining unhybridized and non-specifically hybridized DNA sample. XC1 and XC2 are added to condition the BeadChip surface for the extension reaction. TEM reagents are dispensed into the Flow-Through Chambers to perform singlebase extension of primers hybridized to DNA on the BeadChip. This reaction incorporates labeled nucleotides into the extended primers. 95% formamide/1 mM EDTA is added to remove the hybridized DNA. After neutralization using the XC3 reagent, the labeled extended primers undergo a multi-layer staining process on the Chamber Rack. Next, the Flow-Through

Chambers are disassembled. The BeadChips are washed in the PB1 reagent, and then coated with XC4 reagent and dried.

Item

RA1

XC1

XC2

TEM

XC3

Quantity

(Per 4 BeadChips)

Storage

10 ml (see Setup for special instructions)

-20°C

1 tube -20°C

1 tube

1 tube

Bottle (49 ml)

-20°C

-20°C

Room temperature

Supplied By

Illumina

Illumina

Illumina

Illumina

Illumina

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 139

Item

Quantity

(Per 4 BeadChips)

Storage

LTM (Make sure that all LTM tubes indicate the same stain temperature on the label)

1 tube -20°C

PB1

XC4

Alconox Powder Detergent

EtOH

95% formamide/1 mM EDTA

Bottle (310 ml)

Bottle (310 ml) as needed as needed

30 ml

Supplied By

Illumina

Room temperature

-20°C

Illumina

Illumina

Illumina

Illumina

Illumina Room temperature

-20°C User

WARNING

This protocol involves the use of an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region. Refer to the Infinium Assay

MSDS on your Documentation CD for complete information.

Preparation

`

RA1 is shipped and stored at -20°C. Gradually warm the RA1 reagent to room temperature (22°C), preferably in a 20–25°C water bath. Gently mix to dissolve any crystals that may be present.

`

Thaw all reagent tubes to room temperature (22°C). Centrifuge the thawed reagents to 3000 xg for 1 minute.

`

On the lab tracking worksheet, record:

• Date/Time

• Operator

• Robot

• RA1 barcode

• XC3 barcode

• XC1 barcode(s)

• XC2 barcode(s)

• TEM barcode(s)

• LTM barcode(s)

• ATM barcode(s)

• PB1 barcode

• XC4 barcode(s)

Infinium II Assay Two-Sample HC Protocols

140 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

NOTE

You can print copies of the lab tracking worksheet from the

Documentation CD you received with your system.

Set Up the Chamber Rack

1. Ensure the water circulator reservoir is filled with water to the appropriate level. See the

VWR Operator’s Manual,

VWR part # 110-229.

2. Turn on the water circulator and set it to a temperature that brings the

Chamber Rack to 44°C at equilibrium.

This temperature may vary depending on facility ambient conditions.

(Blowup)

Water Circulator with

Programmable Temperature

Control

Reservoir Cover

Chamber Rack on Robot Bed

Figure 136 Water Circulator Connected to Chamber Rack

3. The temperature displayed on the water circulator LCD screen may differ from the actual temperature on the Chamber Rack. Confirm this using the temperature probe for the Chamber Rack.

4. Remove the bubbles trapped in the Chamber Rack. You must do this every time you run this process. Follow instructions in the

Te-Flow (Tecan

Flow-Through Module) Operating Manual

, Tecan Doc ID 391584.

5. Use the Illumina Temperature Probe in several locations to ensure that

the Chamber Rack is at 44°C (Figure 137).

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 141

Do not leave the temperature probe in the first three rows of the Chamber Rack. Reserve this space for BeadChips.

Temperature Probe

Temperature Probe in Chamber Rack

Figure 137 Illumina Temperature Probe in Chamber Rack

6. For accurate temperature measurement, ensure the Temperature Probe is touching the base of the Chamber Rack.

Prepare the Robot

For instructions on preparing the robot for use in a protocol, and ensuring that the Chamber Rack is properly installed on the post-amplification robot bed, see the Infinium II Assay Lab Setup and Procedures Guide.

Infinium II Assay Two-Sample HC Protocols

142 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

ATM

LTM

TEM

XC2

XC1

Refer to Figure 138 throughout this protocol.

95% Formamide/1 mM EDTA in

Quarter Reservoir

RA1 in Half

Reservoir

XC3 in Full

Reservoir

24 BeadChips in

Chamber Rack

Figure 138 Tecan Eight-Tip Robot (XStain BC2 Setup)

Steps Single-Base Extension and Stain (XStain)

CAUTION

The remaining steps must be performed without interruption.

1. Slide the Chamber Rack into column 36 on the robot bed. Ensure that it is seated properly.

2. At the robot PC, select Infinium II XStain Tasks | XStain BC2.

3. In the Basic Run Parameters pane, enter the Number of BeadChips (up to 24).

The robot PC updates the Required Run Item(s) and the bed map to show the correct position of items on the robot bed.

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 143

Figure 139 XStain BC2 Screen

4. Place a quarter reservoir in the reservoir frame according to the robot

bed map (Figure 139), and add 95% formamide/1 mM EDTA as follows:

• 4 BeadChips: 15 ml

• 24 BeadChips: 25 ml

5. Place a half reservoir in the reservoir frame according to the robot bed map, and add RA1 as follows:

• 4 BeadChips: 10 ml

• 24 BeadChips: 30 ml

6. Place a full reservoir in the reservoir frame according to the robot bed map, and add XC3 as follows:

• 4 BeadChips: 49 ml

• 24 BeadChips: 145 ml

7. Place the XC1, XC2, TEM, LTM, and ATM tubes in the robot tube rack according to the robot bed map. Remove the caps.

8. Make sure that all items are placed properly on the robot bed, that all caps and seals have been removed, and that all the barcodes face to the right.

Start the Robot

1. At the robot PC: a. If you are not running Infinium LIMS, clear the Use Barcodes check box. b. Click Run to start the process. c.

Log in if prompted.

Infinium II Assay Two-Sample HC Protocols

144 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

d. When prompted (Figure 140), enter the stain temperature. The

correct temperature is indicated on the LTM tube. If no temperature is listed, enter 37ºC.

Figure 140 Entering Stain Temperature

e. Observe the robot start to run to ensure that there are no problems.

2. When prompted (Figure 141), wait for the Chamber Rack to reach 44°C.

Do not load the BeadChips or click OK yet.

Figure 141 Adjusting Chamber Rack to 44°C Message

3. When the temperature probe registers 44°C, click OK.

4. When prompted (Figure 142), load the BeadChips and click OK.

Figure 142 Load BeadChips Message

5. Quickly place each Flow-Through Chamber into the Chamber Rack according to the robot bed map.

6. Ensure each Flow-Through Chamber is properly seated to allow adequate heat exchange between the rack and the chamber.

7. On the lab tracking worksheet, record the chamber rack position for each

BeadChip.

8. At the robot PC, click OK. A series of reactions begins, each with a wait time. Message boxes on the robot PC tell you which reaction is occurring and how long the wait time is. The total wait time is 1 hour and

25 minutes.

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 145

9

10

11

12

6

7

8

13

14

15

16

Table 14 List of Reactions

2

3

#

1

4

5

LTM

XC3

ATM

XC3

LTM

XC3

ATM

XC3

LTM

XC3

Reagent Wait Time

RA1 3 minutes

XC1

XC2

10 minutes

10 minutes

TEM 15 minutes

Formamide/

EDTA

7 minutes

XC3 2 minutes

10 minutes

7 minutes

10 minutes

7 minutes

10 minutes

7 minutes

10 minutes

7 minutes

10 minutes

7 minutes

9. When prompted, immediately remove the Flow-Through Chambers from the Chamber Rack. Place them horizontally on the lab bench at room temperature (22°C).

The robot PC sounds an alert and displays a message when the process is complete.

10. Click OK to finish the process.

Verify Reagents and BeadChips for Coating (Infinium

LIMS only)

1. In the Infinium LIMS left sidebar, click Infinium II HC Multi-Sample |

Coat BC2.

2. Scan the barcode(s) of the PB1.

3. Scan the barcode(s) of the XC4.

4. Scan the BeadChip barcodes.

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146 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

5. Click Verify.

Figure 143 (Infinium LIMS) Verifying Reagents and BeadChips for Coating

6. If the reagents are correct and the BeadChips are queued for coating, a blue confirmation message appears at the top of the window. Proceed to the wash and coat step.

7. If any of the reagents are invalid, check the reagent type before rescanning. The reagent name (e.g., PB1) appears at the end of the barcode. Make sure to scan the correct reagent into each box.

8. If any of the BeadChips are not queued for coating, a red error message appears at the top of the window. The error message indicates the first incorrect barcode it finds. Do not proceed with coating. Instead, follow these steps to troubleshoot the problem: a. Click the Reports tab in the upper-right corner.

b. In the left sidebar, click Tracking Reports | Get Queue Status. c.

Scan the BeadChip barcode that appeared in the error message and click Go. d. Note what step the BeadChip is queued for, and proceed with that step.

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 147

Preparing Wash Dishes and Tube Racks

Follow these best practices to optimize the wash and coat process.

`

Take the utmost care to minimize the chance of lint or dust entering the wash dishes, which could transfer to the BeadChips. Place wash dish covers on wash dishes when stored or not in use. Clean wash dishes with low-pressure air to remove particulates before use.

`

In preparation for XC4 BeadChip coating, wash tube racks and wash dishes thoroughly before and after use. Rinse with DI water. Immediately following wash, place racks and wash dishes upside down on a wash rack to dry.

`

Place three layers of Kimwipes on the lab bench. Place a tube rack on top of these Kimwipe layers (do not place on absorbent lab diapers). The staining rack containing BeadChips will be placed on this tube rack after you remove it from the XC4 wash dish.

`

Prepare an additional clean tube rack (Illumina-provided from VWR catalog # 60916-748, must fit internal dimensions of vacuum desiccator) for removal of the BeadChips, one rack per 8 BeadChips. No Kimwipes are required under this tube rack.

Wash and Coat 4 BeadChips

1. Lay out the following equipment on the lab bench:

• 1 staining rack

• 1 vacuum desiccator

• 1 tube rack

• Self-locking tweezers

• Large Kimwipes

• Vacuum hose

2. Set up two top-loading wash dishes, labeled as shown in Figure 144.

3. To indicate the fill volume before filling wash dishes with PB1 and XC4, pour 310 ml water into the wash dishes and mark the water level on the side. Empty the water from the wash dish. This enables you to pour reagent directly from the PB1 and XC4 bottles into the wash dishes, minimizing contaminant transfer from labware to wash dishes.

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Two-Sample HC BeadChip Automated Protocol

Wash Dishes

Staining Rack

Figure 144 PB1 & XC4 Wash Dishes with Staining Rack

4. Pour 310 ml PB1 into the wash dish labeled “PB1.”

5. Submerge the unloaded staining rack into the wash dish with the locking arms and tab facing towards

you (Figure 145). This orients the staining

rack so that you can safely remove the BeadChips.

Locking Arms

Tab

Figure 145 Staining Rack Locking Arms and Tabs

CAUTION

If the staining rack handle is not correctly oriented, the

BeadChips may be damaged when you remove the staining rack handle before removing the BeadChips.

Let the staining rack sit in the wash dish. You will use it to carry the Bead-

Chips after disassembling the Flow-Through Chambers.

6. One at a time, disassemble each Flow-Through Chamber:

a. Using the dismantling tool, remove the two metal clamps (Figure

146).

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 149

CAUTION

It is important to use the dismantling tool to avoid chipping the glass back plates.

Figure 146 Removing Metal Clamps from Flow-Through Chamber

b. Remove the glass back plate.

c.

Set the glass back plates aside. When you finish the XStain BC2 protocol, clean the glass back plates as described in the Infinium II

Assay Lab Setup and Procedures Guide.

d. Remove the spacer.

e. Remove the BeadChip.

CAUTION

Do not touch the face of the BeadChips. Handle them by the barcode end or by the edges.

7. Place BeadChips in the staining rack while it is submerged in PB1. Put all four BeadChips above the staining rack handle. The BeadChip barcodes should face away from you, while the locking arms on the handle face

towards you.

If necessary, briefly lift the staining rack out of the wash dish to seat the

BeadChip. Replace it immediately after inserting the BeadChip.

8. Ensure that the BeadChips are completely submerged.

CAUTION

Do not allow the BeadChips to dry. Submerge each

BeadChip in the wash dish as soon as possible.

9. Move the staining rack up and down 10 times, breaking the surface of

the PB1 (Figure 147).

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150 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Figure 147 Washing BeadChips in PB1

NOTE

If the top edges of the BeadChips begin to touch during either PB1 or XC4 washes, gently move the staining rack back and forth to separate the slides. It is important for the solution to circulate freely between all BeadChips.

10. Allow the BeadChips to soak for an additional 5 minutes.

NOTE

Do not leave the BeadChips submerged in PB1 for longer than 30 minutes.

11. Pour 310 ml XC4 into the dish labeled “XC4,” and cover the dish to prevent any lint or dust from falling into the solution.

Use the XC4 within 10 minutes after filling the wash dish.

NOTE

12. Remove the staining rack from the dish containing PB1 and place it

directly into the wash dish containing XC4 (Figure 148). The barcode

labels on the BeadChips must face away from you, while the locking arms on the handle face towards you, for proper handling and coating.

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 151

Figure 148 Moving BeadChips from PB1 to XC4

13. Slowly move the staining rack up and down 10 times, breaking the surface of the reagent.

NOTE

If the top edges of the BeadChips begin to touch during either PB1 or XC4 washes, gently move the staining rack back and forth to separate the slides. It is important for the solution to circulate freely between all BeadChips.

CAUTION

Use XC4 only once. To process subsequent BeadChips, use a new, clean wash dish with fresh XC4.

14. Prepare a clean tube rack for the staining rack by placing two folded

Kimwipes under the tube rack.

15. Prepare one additional tube rack (Illumina-provided from VWR catalog #

60916-748) that fits the internal dimensions of the vacuum desiccator.

16. Remove the staining rack in one smooth, rapid motion and place it directly on the prepared tube rack, making sure the barcodes face up and the locking arms and tab face

down

(Figure 149, showing

8 BeadChips).

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152 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Figure 149 Staining Rack in Correct Orientation

17. To ensure uniform coating, place the staining rack on the center of the tube rack, avoiding the raised edges.

Figure 150 Moving BeadChip Carrier from XC4 to Tube Rack

18. For each of the four BeadChips, working top to bottom: a. Continuing to hold the staining rack handle, carefully grip each

BeadChip at its barcode end with self-locking tweezers.

NOTE

The XC4 coat is slippery and makes the BeadChips difficult to hold. The self-locking tweezers grip the BeadChip firmly and help prevent damage.

b. Place the BeadChip horizontally on a tube rack with the barcode

facing up and towards you (Figure 151).

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 153

Figure 151 Placing BeadChips on Tube Rack

CAUTION

To prevent wicking and uneven drying, do not allow the

BeadChips to rest on the edge of the tube rack or to touch each other while drying.

19. Holding the top of the staining rack in position, grasp the handle between your thumb and forefinger. Push the tab up and push the

handle away from you to unlock it. Pull up the handle and remove (Figure

152).

Tab

Handle

Figure 152 Removing Staining Rack Handle

20. Dry the BeadChips:

a. Place the tube rack with the BeadChips (Figure 160) into the

desiccator. Check the vacuum pressure and make sure that the valve is securely attached.

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154 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol b. Start the vacuum, using at least 508 mm Hg (0.68 bar).

c.

To ensure that the dessicator is properly sealed, gently lift the lid of the vacuum desiccator. It should not lift off the desiccator base.

d. Dry under vacuum for 50–55 minutes.

Drying times may vary according to room temperature and humidity.

21. Proceed to step 36, page 161.

Wash and Coat

24 BeadChips

Equipment Needed

`

1 staining rack

`

3 vacuum desiccators (1 per 8 samples)

`

3 tube racks (1 per 8 samples)

`

Self-locking tweezers

`

Large Kimwipes

`

Vacuum hose

1. Set up two top-loading wash dishes, labeled as shown in Figure 153.

2. To indicate the fill volume before filling wash dishes with PB1 and XC4, pour 285 ml water into the wash dishes and mark the water level on the side. Empty the water from the wash dish. This enables you to pour reagent directly from the PB1 and XC4 bottles into the wash dishes, minimizing contaminant transfer from labware to wash dishes.

Wash Dishes

Staining Rack

Figure 153 PB1 and XC4 Wash Dishes with

Staining

Rack

3. Pour 285 ml PB1 into the wash dish labeled “PB1.”

4. Submerge the unloaded staining rack into the wash dish with the locking

arms and tab facing you (Figure 154). This orients the staining rack so

that you can safely remove the BeadChips.

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 155

Locking Arms

Tab

Figure 154 Staining Rack Locking Arms and Tabs

CAUTION

If the staining rack handle is not correctly oriented, the

BeadChips may be damaged when you remove the staining rack handle before removing the BeadChips.

5. Let the staining rack sit in the wash dish. You will use it to carry the

BeadChips after disassembling the Flow-Through Chambers.

6. One at a time, disassemble each Flow-Through Chamber:

a. Using the dismantling tool, remove the two metal clamps (Figure

155).

CAUTION

It is important to use the dismantling tool to avoid chipping the glass back plates.

Figure 155 Removing Metal Clamps from Flow-Through Chamber

b. Remove the glass back plate.

c.

Set the glass back plates aside. When you finish the XStain BC2 protocol, clean the glass back plates as described in the Infinium II

Assay Lab Setup and Procedures Guide.

d. Remove the spacer.

e. Remove the BeadChip.

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156 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

CAUTION

Do not touch the face of the BeadChips. Handle them by the barcode end or by the edges.

7. Place BeadChips in the staining rack while it is submerged in PB1. Place

12 BeadChips above the handle and 12 below. The BeadChip barcodes should face away from you, while the locking arms and tab face towards you.

If necessary, briefly lift the staining rack out of the wash dish to seat the

BeadChip. Replace it immediately after inserting the BeadChip.

8. Ensure that the BeadChips are completely submerged.

CAUTION

Do not allow the BeadChips to dry. Submerge each

BeadChip in the wash dish as soon as possible.

9. Move the staining rack up and down 10 times, breaking the surface of

the PB1 (Figure 156).

NOTE

If the top edges of the BeadChips begin to touch during either PB1 or XC4 washes, gently move the staining rack back and forth to separate the slides. It is important for the solution to circulate freely between all BeadChips.

10. Allow the BeadChips to soak for an additional 5 minutes.

Figure 156 Washing BeadChips in PB1

NOTE

Do not leave the BeadChips submerged in PB1 for longer than 30 minutes.

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 157

11. Pour 285 ml XC4 into the dish labeled “XC4,” and cover the dish to prevent any lint or dust from falling into the solution. Place the bottle with excess XC4 in a readily available location for topping off the XC4 wash dish during the coating procedure.

Use the XC4 within 10 minutes after filling the wash dish.

NOTE

12. Remove the staining rack from the dish containing PB1 and place it

directly into the wash dish containing XC4 (Figure 157). The barcode

labels on the BeadChips must face away from you, while the locking arms on the handle face towards you, for proper handling and coating.

13. Move the staining rack up and down 10 times, breaking the surface of the XC4.

Figure 157 Moving BeadChips from PB1 to XC4

NOTE

If the top edges of the BeadChips begin to touch during either PB1 or XC4 washes, gently move the staining rack back and forth to separate the slides. It is important for the solution to circulate freely between all BeadChips.

14. Allow the BeadChips to soak for an additional 5 minutes.

CAUTION

Use XC4 only once. To process subsequent BeadChips, use a new, clean wash dish with fresh XC4.

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158 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

15. Prepare a clean tube rack for the staining rack by placing two folded

Kimwipes under the tube rack.

16. Prepare one additional tube rack per 8 BeadChips (Illumina-provided from VWR catalog # 60916-748) that fits the internal dimensions of the vacuum desiccator.

17. Remove the staining rack in one smooth, rapid motion and place it directly on the prepared tube rack, making sure the barcodes face up

and the locking arms and tab face down (Figure 159).

Figure 158 Staining Rack in Correct Orientation

18. To ensure uniform coating, place the staining rack on the center of the tube rack, avoiding the raised edges.

Figure 159 Moving BeadChip Carrier from XC4 to Tube Rack

19. For the first eight BeadChips, working top to bottom: a. Continuing to hold the staining rack handle, carefully grip each

BeadChip at its barcode end with self-locking tweezers.

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 159

NOTE

The XC4 coat is slippery and makes the BeadChips difficult to hold. The self-locking tweezers grip the BeadChip firmly and help prevent damage.

b. Put the eight BeadChips on the tube rack as shown in Figure 160,

with six BeadChips on top of the rack and two BeadChips on the bottom. The barcode ends should be towards you, and the

BeadChips should be completely horizontal.

CAUTION

To prevent wicking and uneven drying, do not allow the

BeadChips to rest on the edge of the tube rack or to touch each other while drying.

Figure 160 Placing BeadChips on Tube Rack

20. Return the staining rack to the XC4 wash dish and top off the dish until the BeadChips are completely covered with remaining XC4 reagent.

21. Soak the BeadChips for 10 seconds.

22. Dry the first 8 BeadChips:

a. Place the tube rack with the first 8 BeadChips (Figure 160) into the

desiccator. Check the vacuum pressure and make sure that the valve is securely attached.

b. Start the vacuum, using at least 508 mm Hg (0.68 bar).

c.

To ensure that the dessicator is properly sealed, gently lift the lid of the vacuum desiccator. It should not lift off the desiccator base.

d. Dry under vacuum for 50–55 minutes.

Drying times may vary according to room temperature and humidity.

23. Remove the staining rack with the remaining BeadChips in one rapid motion from the XC4 wash dish and place it directly on the tube rack.

Ensure that the BeadChips are horizontal with the barcodes facing up.

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160 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

Figure 161 Moving BeadChip Carrier from XC4 to Tube Rack

24. Remove the four BeadChips that remain above the staining rack handle and place them on the tube rack.

25. Holding the top of the staining rack in position, grasp the handle between your thumb and forefinger. Push the tab up with your thumb and push the handle away from you to unlock it. Pull up the handle and

remove (Figure 162).

Tab

Handle

Figure 162 Removing Staining Rack Handle

26. Place BeadChips on the tube rack as shown in Figure 160 until there are

six BeadChips on top of the rack and two BeadChips on the bottom. The barcode ends should be towards you, and the BeadChips should be completely horizontal.

Part # 11280749 Rev. A

Single-Base Extension & Stain BC2 161

27. Return the staining rack with the last 8 BeadChips to the XC4 wash dish and top off the wash dish until BeadChips are completely covered with remaining XC4 reagent.

28. Soak BeadChips for 10 seconds.

29. Dry the second set of 8 BeadChips:

a. Place the tube rack with the second set of 8 BeadChips (Figure 160)

into the desiccator. Check the vacuum pressure and make sure that the valve is securely attached.

b. Start the vacuum, using at least 508 mm Hg (0.68 bar).

c.

To ensure that the dessicator is properly sealed, gently lift the lid of the vacuum desiccator. It should not lift off the desiccator base.

d. Dry under vacuum for 50–55 minutes.

30. Remove staining rack with the remaining 8 BeadChips in one rapid motion from the XC4 wash dish and place it directly on tube rack. Ensure that the BeadChips are horizontal with the barcodes facing up.

31. Place BeadChips on the tube rack as shown in Figure 160 until there are

six BeadChips on top of the rack and two BeadChips on the bottom. The barcode ends should be towards you, and the BeadChips should be completely horizontal.

32. Place the tube rack with the third set of 8 BeadChips (Figure 160) into

the desiccator. Check the vacuum pressure and make sure that the valve is securely attached.

33. Start the vacuum, using at least 508 mm Hg (0.68 bar).

34. To ensure that the dessicator is properly sealed, gently lift the lid of the vacuum desiccator. It should not lift off the desiccator base.

Figure 163 Testing Vacuum Seal

35. Dry under vacuum for 50–55 minutes.

36. Release the vacuum by turning the handle very slowly.

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162 CHAPTER 3

Two-Sample HC BeadChip Automated Protocol

WARNING

Air should enter the desiccator very slowly to avoid disturbing the contents. Improper use of the vacuum desiccator can result in damage to the BeadChips. This is especially true if you remove the valve plug while a vacuum is applied. For detailed vacuum desiccator instructions, see the documentation included with the desiccator.

37. Store the desiccator with the red valve plug in the desiccator’s three-way valve to stop accumulation of dust and lint within the valve port. Remove the red plug from the three-way valve before applying vacuum pressure.

38. Touch the borders of the chips (do not touch the stripes) to ensure that the etched, bar-coded side of the BeadChips are dry to the touch.

39. If the underside feels tacky, manually clean the underside of the

BeadChip to remove any excess XC4. The bottom two BeadChips are the most likely to have some excess.

a. Hold the BeadChip at a downward angle to prevent excess EtOH from dripping from the wipe onto the stripes.

a. Wrap a pre-saturated Prostat EtOH Wipe around your index finger.

b. Wipe along the underside of the BeadChip five or six times, until the surface is clean and smooth.

Do not touch the stripes.

CAUTION

40. If you are using Infinium LIMS: a. In the Infinium LIMS left sidebar, click Infinium II HC Multi-Sample |

Coat BC2. b. Scan the reagent barcodes and BeadChip barcodes and click Save.

Infinium LIMS records the data and queues the BeadChips for the

next step, Image BC2.

41. Clean the glass back plates. For instructions, see the Infinium II Assay Lab

Setup and Procedures Guide.

42. Clean the Hyb Chambers: a. Remove the rubber gaskets from the Hyb Chambers.

b. Rinse all Hyb Chamber components with DI water.

c.

Thoroughly rinse the humidifying buffer reservoirs.

43. Discard unused reagents in accordance with facility standards.

Part # 11280749 Rev. A

Image BC2 163

Image BC2

The BeadChips are now ready for scanning. For instructions, see the Infinium

II Assay Lab Setup and Procedures Guide. Image the BeadChips within

72 hours.

•

Infinium II Assay Two-Sample HC Protocols

Illumina, Inc.

9885 Towne Centre Drive

San Diego, CA 92121-1975

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America) [email protected]

www.illumina.com