iPLEX HS Colon and Lung Panels USG-CUS-084 R01 › wp-content › uploads › 2017 › 05 › ...4...

For Research Use Only. Not for Use in Diagnostic Procedures.

iPLEX® HS Colon and Lung Panels USER GUIDE

iPLEX® HS Colon and Lung Panels User GuideDOC. USG-CUS-084 R014/3/17

Copyright 2017. All rights reserved. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, or stored in a database or retrieval system, for any reason other than a licensee's internal use, without the prior written permission of Agena Bioscience, Inc. Printed in the United States of America.

TRADEMARKSMassARRAY, iPLEX, and SpectroCHIP are registered trademarks of Agena Bioscience, Inc. Agena Bioscience is a trademark of Agena Bioscience, Inc. All other trademarks or service marks set forth herein are the property of their respective owners.

PATENTS

Agena Bioscience’s patented nucleic acid analysis by mass spectrometry methods and products are protected under United States patent rights including but not limited to; 6,440,705; 6,558,623; 6,730,517; 6,979,425; 6,994,969; 7,019,288; 7,025,933; 7,332,275; 7,390,672; 7,501,251; 7,888,127; 7,917,301; 8,003,317; 8,315,805; 8,349,566; 9,249,456; and 9,310,378, and patents pending including but not limited to US20130017960, and foreign counterparts including but not limited to EP1173622B1, EP1727911B1, EP1546385B1, EP1332000B1, EP1613723B1, EP1660680B1, and EP2107129B1.

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Contents

Chapter 1 Overview and Inventory Checklist . . . . . . . . . . . . . . . .3

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . 3

Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . 3

Inventory Checklist . . . . . . . . . . . . . . . . . . . . . . . . 4

Laboratory Work Areas . . . . . . . . . . . . . . . . . . . . . .8

Important Procedures and Guidelines. . . . . . . . . . . . . . . . . 8

Customer Support . . . . . . . . . . . . . . . . . . . . . . . .9

Chapter 2 Setting up the Experiment in the Software . . . . . . . . . . . 11

Importing the Assay Design File with MassARRAY Typer AssayEditor . . . . . . .11

Creating a Virtual Plate in MassARRAY Typer PlateEditor . . . . . . . . . . 11

Chapter 3 Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . 15

Preparing DNA from Samples . . . . . . . . . . . . . . . . . . . 15

Performing PCR Amplification . . . . . . . . . . . . . . . . . . . 16

Performing SAP Treatment . . . . . . . . . . . . . . . . . . . . 17

Performing the iPLEX HS Extension Reaction . . . . . . . . . . . . . . 19

Next Steps: Processing Options. . . . . . . . . . . . . . . . . . . 21

Chapter 4 MassARRAY Nanodispenser and MassARRAY Analyzer Processing(96- or 384-format) . . . . . . . . . . . . . . . . . . . . . 23

Desalting the iPLEX HS Extension Reaction Product (Dry Resin Method) . . . . . 23

Dispensing Samples to a SpectroCHIP Array (MassARRAY Nanodispenser) . . . . 24

Acquiring Data on the MassARRAY Analyzer . . . . . . . . . . . . . . 26

Chapter 5 MassARRAY System with Chip prep module Processing(96-format) . . . . . . . . . . . . . . . . . . . . . . . . . 31

Preparing the Plate . . . . . . . . . . . . . . . . . . . . . . . 31

Creating an Input File . . . . . . . . . . . . . . . . . . . . . . 31

Starting the Software and Checking Instrument Status . . . . . . . . . . . 32

Loading the Deck . . . . . . . . . . . . . . . . . . . . . . . 33

Setting Up the Run in the Software 3 . . . . . . . . . . . . . . . . . . 4

Starting the Automatic Run . . . . . . . . . . . . . . . . . . . . 36

Removing Plates and Calibrant . . . . . . . . . . . . . . . . . . . 36

Chapter 6 Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . 37

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Generating Reports in TyperAnalyzer . . . . . . . . . . . . . . . . 38

Reading the iPLEX HS Report . . . . . . . . . . . . . . . . . . . 40

USG-CUS-084 R01iPLEX® HS Colon and Lung Panel User GuideFor Research Use Only. Not for use in diagnostic procedures.

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Appendix A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . 49

Appendix B Quick Reference Guide: Reaction Conditions . . . . . . . . . . 55

PCR Reaction Conditions . . . . . . . . . . . . . . . . . . . . 55

SAP Reaction Conditions . . . . . . . . . . . . . . . . . . . . . 55

iPLEX HS Extension Reaction Conditions . . . . . . . . . . . . . . . . 56

Appendix C Additional Peaks . . . . . . . . . . . . . . . . . . . . . . .57

Appendix D iPLEX HS Lung Panel Assays . . . . . . . . . . . . . . . . . 59

Appendix E Licensing . . . . . . . . . . . . . . . . . . . . . . . . . 63

Appendix F Warranty . . . . . . . . . . . . . . . . . . . . . . . . . .67


Chapter 1

Overview and Inventory Checklist

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Inventory Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Laboratory Work Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Important Procedures and Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Customer Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1.1 Introduction

The iPLEX® HS Colon Panel and iPLEX HS Lung Panel, for use on the MassARRAY® System, enable mutation detection as low as 1% allele frequency from poor quality and degraded samples such as FFPE tissue, FNA, and cytology blocks.

Table 1.1 iPLEX HS Colon Panel Content

Table 1.2 iPLEX HS Lung Panel Content

The iPLEX HS Colon and Lung Panels and the MassARRAY System are for research use only and not for use in diagnostic procedures.

1.2 Workflow Overview

The iPLEX HS Colon and Lung Panels use PCR to amplify target regions of interest. After the removal of unincorporated dNTPs, a sequence-specific primer extension step is performed using the supplied Extend primers and iPLEX Reagents. After sample analysis and data acquisition on the MassARRAY Analyzer, genotype reports are generated with MassARRAY Typer software.

Gene # of Mutations Coverage

BRAF 3 Codon 469 of exon 11; codons 594, 600 of exon 15

EGFR 2 Extracellular domain mutations across exon 12

KRAS 47 Codons 12, 13 of exon 2; codons 59, 61 of exon 3; codons 117, 146 of exon 4

NRAS 30 Codons 12, 13 of exon 2; codons 59, 61 of exon 3; codons 117, 146 of exon 4

PIK3CA 4 Codons 542, 545 of exon 9; codon 1047 of exon 20

Gene # of Mutations Coverage

BRAF 4 Codon 469 of exon 11; codons 594, 600 of exon 15

EGFR 46 Exon 19 indels, exon 20 insertions, and substitutions across exons 18, 19, 20, and 21

ERBB2 2 Exon 20 insertions

KRAS 14 Codons 12, 13 of exon 2; codon 61 of exon 3

PIK3CA 4 Codons 542, 545 of exon 9; codon 1047 of exon 20


Chapter 1 Overview and Inventory Checklist4

Workflow steps are shown in Table 1.3.

Table 1.3 Workflow Steps

1.3 Inventory Checklist

Kit Contents Table 1.4 lists the required Agena Bioscience reagents needed to run the iPLEX HS Colon and Lung Panels. The panels may be run in 96- or 384-well formats. Both kits allow testing of up to 120 samples.

Upon receipt, store the items as described in Table 1.4.

Table 1.4 iPLEX HS Colon and Lung Panel Reagents

Software Setup See...

Import the assay design file into MassARRAY Typer Assay Editor software.Only required before the first time you run the panel.

Section 2.1

Create a virtual plate in MassARRAY Typer Plate Editor software. Section 2.2

Assay Protocol See...

Prepare DNA from samples. Section 3.1

Amplify DNA using the global PCR primer. Section 3.2

Perform SAP treatment to dephosphorylate any remaining free dNTPs. Section 3.3

Process the extension reactions using the Extend primers. Section 3.4

Data Acquisition and Analysis See...

Desalt the extension products, transfer to a SpectroCHIP Array, and analyze on the MassARRAY Analyzer.• With MassARRAY Nanodispenser and MassARRAY Analyzer (96-

and 384-format)• With MassARRAY System with Chip prep module (96-format)

Chapter 4

Chapter 5

View data and generate reports. Chapter 6

Materials Provided10 x 96 KitQuantity

3 x 384 KitQuantity

Shipping Condition

Storage Temperature

Storage Location (see Table 1.8 Lab Areas)

iPLEX HS Colon Panel

or

iPLEX HS Lung Panel• Global PCR Primer• Extension Primers

(EXT W1 - EXT W8)• Termination Mixes

(W1 - W8)

1 1 Dry Ice -10 to - 25°C Lab Area 2

PCR Accessory Set• MgCl2• 10X PCR Buffer• dNTP Mix

1 1 Dry Ice -10 to - 25°C Lab Area 2

PCR Enzyme 1 1 Dry Ice -10 to - 25°C Lab Area 2


Chapter 1 Overview and Inventory Checklist 5

*Because both iPLEX HS and iPLEX Pro chemistries are supported by the iPLEX Pro Reagent Kit, you will receive as part of the set the regular iPLEX Pro Termination Mix. Please note that in order to achieve down to 1% sensitivity, you must use the iPLEX HS Termination Mixes W1 - W8 that are provided with the iPLEX HS Colon or Lung Panel kits.

Required Equipment and Instruments

See MassARRAY System: Recommended Lab Equipment and Set-up, available on the Product Support page of AgenaCx.com, for more detailed information on recommended lab set up and recommended vendors.

Table 1.5 Required Instruments and Equipment

iPLEX Pro Reagent Kit• SAP Buffer• SAP Enzyme• iPLEX Pro Buffer

Plus (10X)• iPLEX Pro Enzyme• iPLEX Pro

Termination Mix*• 3-Pt. Calibrant

1 medium 1 medium Dry Ice -10 to - 25°C Lab Area 2

SpectroCHIP Array and Resin Kit

10 Chips

28 g Clean Resin

3 Chips

9g Clean Resin

Ambient Temperature

Room Temperature

Lab Area 3 (paraffin-cover Clean Resin)

Materials Provided10 x 96 KitQuantity

3 x 384 KitQuantity

Shipping Condition

Storage Temperature

Storage Location (see Table 1.8 Lab Areas)

Instruments and Equipment Specification

MassARRAY Analyzer and

MassARRAY Nanodispenser RS1000

OR

MassARRAY System with Chip prep module

Plate centrifuge Min. RCF: 3200 x g, min. speed: 4000 rpm

Vortex Variable speed, suitable for tubes and plates

Mini tube centrifuge Recommend additional rotor option for 2 x 8-tube PCR strips.

Plate/tube rotator (not required if using MassARRAY System with Chip prep module)

360º rotation, with standard rotisserie

Thermocycler With appropriate plate block



Other Required Reagents and Labware

See MassARRAY System: Recommended Lab Equipment and Set-up, available on the Product Support page of AgenaCx.com, for more detailed information on recommended lab set up and recommended vendors.

Table 1.6 Other Required Reagents and LabwareItem Specifications

Electronic multichannel pipettes and filtered tips (Optional; can use manual multichannel pipettes)

8- or 12-channel electronic adjustable tip spacing pipette; 0.5 μL -12.5 μL

Manual multichannel pipettes and filtered tips 8- or 12-channel pipette0.5 μL -10 μL20 μL - 200 μL

Single channel pipettes and filtered tips 0.1 μL - 2 μL0.5 μL -10 μL10 μL -100 μL100 μL -1000 μL

Microtubes RNase-, DNase-, human DNA-, and PCR inhibitor-free

Volume: 1.5 mL, 0.5 mL

PCR strip tubes (optional) 8- or 12-well strips with caps; volume: 0.2 mL

Tube racks

Sealing roller tool and paddle

Disposable pipetting reservoirs DNase- and RNase-free; volume: 25 mL; sterile

Clear adhesive plate seals Strong adhesive, -20 ºC to 120 ºC

Pressure pads

Molecular grade water Sterile, nuclease- and DEPC-free; >18.2 MΩ; 500 mL bottles

Ethanol (only needed for Nanodispenser RS1000) Absolute (200 proof)

Tween 20 (only needed for Nanodispenser RS1000) Nonionic detergent solution; 10% w/v

DNA AWAY™

Type 1 water (used for supply/rinse water) NCCLS, CAP or ATSM; >18.2 MΩ

Resin dimple plate and scraper (only needed for use with the MassARRAY Nanodispenser RS1000)

Supplied with Agena Bioscience MassARRAY System

For 96-format experiments:

96-well full-skirted, low profile plates

Alternatives: • non-skirted low profile plates

• non-skirted standard plates

• semi-skirted plates

If non-skirted plates are used, check plates for warping after thermocycling.

Users with a MassARRAY System with Nanodispenser in a 96/384 format MUST use low profile plates in the dispensing step.

Max fill vol.: 0.2 mL






Required Software Table 1.7 Required Software

96-well fully-skirted MTP plate base (only needed if using non-skirted plates on the Nanodispenser RS1000)

Agena Bioscience 179108 (included with Nanodispenser RS1000)

For 384-format experiments:

96-well full-skirted, low profile plates

Alternatives: • non-skirted low profile plates

• non-skirted standard plates

• semi-skirted plates





384-well full-skirted microtiter plates Working vol.: 25 μL; max vol.: 40 μL

Item Specifications

Software Supplier

MassARRAY Typer 4.0.163 or higher• Assay Editor. Imports assay design

files to the MassARRAY database. See page 11.

• PlateEditor. Creates a virtual plate that represents the physical plate with the analytes (sample plate). See page 11

• TyperAnalyzer. Displays the spectral results data in table and graph formats and generates results reports. See page 37.

Agena Bioscience (download from the Product Support page on AgenaCx.com)

’R’ Environment, including RODBC, doBy, gap, and RSQLite packages)

http://cran.r-project.org

See MassARRAY Typer Release Notes on the Product Support page on AgenaCx.com for instructions for downloading the R software and installing the necessary packages.



Some MassARRAY Typer reports are generated in both CVS and HTML format. Internet Explorer (9, 10, or 10 Compatibility View), Firefox, or Chrome Internet browsers are required to view the HTML reports.

1.4 Laboratory Work Areas

The laboratory space should include three separate (non-contiguous) work areas to prevent contamination of PCR products. Table 1.8 shows the activities that are conducted in each area.

Table 1.8 Lab Area Activities

1.5 Important Procedures and Guidelines

It is important that the following procedures be adhered to throughout processing of iPLEX reactions.

• Follow Good Laboratory Practices.

MassARRAY Analyzer software• Chip Linker. Links the virtual plate

created in MassARRAY Typer Plate Editor with the associated SpectroCHIP Array. See page 27, or page 31.

• SpectroACQUIRE. Provides the graphical user interface. Enables user to set up and start an automatic run or perform manual data acquisition. See page 29, or page 34.

• AnalyzerControl. Enables communication between the MassARRAY Analyzer and the SpectroACQUIRE software.

• MassARRAY Caller. Analyzes the data as it is acquired. Identifies and calls the spectra peaks. Exports the data to the MassARRAY database when the automatic run is completed.

• Chip prep control. Controls the Chip prep module.

Agena Bioscience (part of MassARRAY Analyzer and MassARRAY System with Chip prep module)

Software Supplier

Lab Area Activities

1 Isolation and dilution of DNA and preparation of the DNA source plate.

2

Pre-PCR preparation, including preparation of the DNA reaction plate, preparation of the PCR cocktail, and addition of the PCR cocktail to the reaction plate. Preparation of the SAP and extension cocktails.

3

Thermocycling the reaction plate after addition of PCR cocktail, addition of the SAP cocktail to the reaction plate and thermocycling, addition of the extension reaction cocktail to the reaction plate and thermocycling, desalting, nanodispensing, and data acquisition.



• Wear gloves and safety glasses when handling all equipment, components, and reagents. Be careful not to contaminate samples, reagents, mixes, and/or pipette tips.

• When preparing a cocktail or solution, add reagents in the order in which they are listed in the table.

• When running more than one reaction, calculate and apply an appropriate overhang to reagent volumes.

• Vortex and centrifuge tubes and plates with reagents before use or cycling.

• Seal plates with adhesive PCR sealer when not in use. Ensure proper sealing to avoid evaporation.

• When programming the thermocycler for the first time in each step, save and name the programs (for example, PCR, SAP, iPLEX). Then for future reactions, just run the appropriate program.

• Store the reagents (stocks, dilutions, etc.) and finished cocktails in plates at -20°C when not in use.

• Store DNA samples at -20°C when not in use.

• Store DNA samples at 4°C when in use.

• Calibrate and optimize instruments following each instrument's corresponding manual before each use.

• Refer to the appropriate Material Safety Data Sheets (MSDS), available on AgenaCx.com, for the safety recommendations on all chemicals used.

• When working in the pre-PCR clean labs (i.e., Lab Area 1 and Lab Area 2) follow proper gowning practices.

• Before performing pre-PCR cycling work, clean the bench areas, pipettes, and hood work area with DNA AWAY or similar solution, followed by 70% ethanol, and illuminate with UV light for 30 minutes.

• When working in the post-PCR lab (i.e., Lab Area 3) always wear gloves and lab coat.

• Clean post-PCR bench areas with DNA AWAY.

1.6 Customer Support

Please contact your local Agena Bioscience office for customer support.

CORPORATE HEADQUARTERS & NORTH AMERICA Agena Bioscience, Inc.4755 Eastgate MallSan Diego, CA 92121 USAPhone: 1-858-882-2800Order Desk: 1-858-202-9301Order Desk Fax: 1-858-202-9220Order Desk Email: [email protected] Desk: 1-877-4-GENOME or (+1) 858-882-9300E-mail: [email protected]



EUROPE Agena Bioscience GmbHMendelssohnstrasse 15DD-22761, HamburgGermanyPhone: (+49) 40-899676-0Fax: (+49) 40-899676-10Order Desk: [email protected]: [email protected]

ASIA PACIFIC Agena Bioscience, Inc.300 Herston RoadHerston, QLD 4006AustraliaPhone: (+61) 7 3088 1600Fax: (+61) 7 3088 1614Email: [email protected]

CHINA Agena Bioscience (Shanghai) Co., Ltd.Room 1609-1613, Building AFenglin International CenterNo. 380 Fenglin RoadXuhui DistrictShanghai 200032PR ChinaPhone: (+86) 21 6427 0566Fax: (+86) 21 6427 0511Email: [email protected]

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Chapter 2

Setting up the Experiment in the Software

Importing the Assay Design File with MassARRAY Typer AssayEditor . . . . . . . . . . . . . . . . 11Creating a Virtual Plate in MassARRAY Typer PlateEditor . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.1 Importing the Assay Design File with MassARRAY Typer AssayEditor

The iPLEX HS Colon or Lung Panel assay design file must be imported into the MassARRAY database, using MassARRAY Typer AssayEditor. The assay design file contains all the essential assay design specifications for genotyping analysis.

The assay design file only needs to be imported once, prior to running your panel for the first time.

The instructions presented here are for users who are familiar with importing assays using MassARRAY Typer AssayEditor. For comprehensive information on using MassARRAY Typer application software, refer to the MassARRAY Typer 4 User Guide, which is available by contacting Agena Bioscience Customer Support or from AgenaCx.com.

To import the assay design file:

1. Download the Assay Design File from the Product Support page on AgenaCx.com.

2. Open MassARRAY Typer AssayEditor.

3. Create a new assay project in the Database Browser by right-clicking the root node and selecting Project Administrator.

4. Add a new Assay Project with an appropriate name. The new Assay Project will appear in the database browser. The Assay Design File will be stored in this project.

5. Right-click on the newly created Assay Project and select Import Assay Group in Designer format...

6. Remove the checkmarks next to Design Summary and SNP Group. Make sure that there is a checkmark next to Assay Group.

7. Click the Browse button next to Assay Group.

8. Navigate to the folder where the assay design file was saved, select it, and click Open.

9. Click the Import button to import the file.

2.2 Creating a Virtual Plate in MassARRAY Typer PlateEditor

Once the assay design file has been imported, a virtual sample plate must be created in the MassARRAY database. This virtual sample plate represents the physical sample plate with the analytes. Use MassARRAY Typer PlateEditor to:


Chapter 2 Setting up the Experiment in the Software12

• Create a sample group and enter it into the database.

• Create a virtual sample plate.

• Assign assays to the plate.

• Assign samples to the plate.

See the MassARRAY Typer 4 User Guide, available by contacting Agena Bioscience Customer Support or from AgenaCx.com, for detailed instructions on creating a plate with PlateEditor.

Creating a Sample Group

1. Create a text file (.txt) of sample descriptions in a spreadsheet application such as Microsoft® Excel. The file must contain the sample ID in column A, and may contain a description in column B.

2. Each sample group belongs to a sample project, and each sample project belongs to a sample customer. Create a sample customer and sample project in the database, if you haven’t already done so.

a. Open the Plate Editor software and click on the Sample tab.

b. To create a sample customer, right-click the root-node and select Add New Sample Customer. Enter a sample customer ID and additional optional information and click OK. The new sample customer will appear in the Sample tab.

c. To create a sample project, right-click the sample customer that the sample project will be under and select Add New Sample Project. Enter a sample project ID and optional additional information and click OK. The new sample project will appear in the Sample tab.

3. Create a sample group.

a. Right-click on the sample project that you want to add the sample group to and select Add New Sample Group.

b. Enter a sample group ID and click the folder button in the toolbar to browse to the location of your sample group text file, and click Open, then click OK.

NOTEIf you use wild-type samples on your reaction plate as baseline data instead of the provided baseline file, designate each wild-type sample on the plate with a common name (e.g., NORM or WT). The software will use all samples with the common name (e.g., 1NORM, 2NORM, etc., or WT_1, WT_2, etc.) as the baseline data. You will specify the common name in the Text label for WT samples field of the Analysis Settings window (see page 38 and Figure 6.2).

Creating a Plate 1. Each plate belongs to a project, and each project belongs to a customer. Create a customer and project in the database, if you haven’t already done so.


Chapter 2 Setting up the Experiment in the Software 13

a. In the Plate Editor software click on the Plate tab.

b. To create a customer, right-click the root-node and select New Customer. Enter a customer ID and additional optional information and click OK. The new customer will appear in the Plate tab.

c. To create a project, right-click the customer that the project will be under and select New Project. Enter a project ID and optional additional information and click OK. The new project will appear in the Plate tab.

2. Create a Plate.

a. Right-click on the project that you want to add the plate to and select New Plate.

b. Enter a plate ID and select the plate type (96- or 384-well) and click OK. The new plate will appear in the Plate tab and a plate layout will be created automatically, based on the plate type specified.

Applying Assays to the Plate

1. In the Plate tab, select the plate that was just created.

2. Select the Assay tab, and locate the assay design file you imported earlier.

3. In the plate layout, select the wells of interest.

4. In the Assay tab, right-click the plex or assay that you want to assign to the selected wells and select Add plex or Add assay.

5. Add the plex “W9” to all wells.

Applying Samples to the Plate

1. Click on the Sample tab.

2. In the plate layout, select the wells of interest.

3. Right-click the sample or sample group that you want to assign to the selected wells and select Apply Samples from Group or Add Sample.

4. Select File > Save from the toolbar.


Chapter 2 Setting up the Experiment in the Software14

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Chapter 3

Assay Protocol

Preparing DNA from Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Performing PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Performing SAP Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Performing the iPLEX HS Extension Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Next Steps: Processing Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

The following protocol is for simultaneous processing of 12 DNA samples tested with all 8 multiplexes using a 96-well plate. Users wishing to run more samples in a 384-well format will perform PCR and SAP amplification in a 96-well plate as shown here, then transfer analyte to a 384-well plate before the iPLEX HS extension reaction. For experienced users, Appendix B provides a quick reference guide with reaction conditions for the PCR, SAP, and extension reactions.

3.1 Preparing DNA from Samples

IMPORTANT!Perform this procedure in Lab Area 1. See MassARRAY System: Recommended Lab Equipment and Set-up, available on the Product Support page of AgenaCx.com, for more detailed information on recommended DNA extraction and quantification methods and kits.

Types of Samples The iPLEX HS Colon and Lung Panels can be performed on genomic DNA or isolated from a variety of samples, including fresh tissue, frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue, cell lines, blood plasma, FNA, core needle biopsies, and cytology cell blocks.

Method You may use any method that you prefer to isolate genomic DNA from your samples. Note that DNA isolated from FFPE samples is usually fragmented and of lower molecular weight than DNA from fresh or frozen samples. The degree of fragmentation depends on the type and age of the sample and the conditions used for fixation. A commercial, lysis-based kit such as Qiagen’s QIAamp DNA FFPE Tissue Kit or a similar product is recommended for processing FFPE samples.

Quality Assessment Use a spectrophotometric or fluorometric method to determine the concentration and relative purity of your genomic DNA.

To produce large quantities of PCR amplicons for use with the MassARRAY System, it is recommended that the genomic DNA template be highly pure. The ratio of ultraviolet (UV) spectrophotometer readings at 260 nm and 280 nm wavelengths should be between 1.7 and 2.0 (i.e., A260/A280 = 1.7-2.0). Ratios in this range indicate the presence of nucleic acid with low amounts of contaminating protein. In general, we have found genomic DNA of this quality is suitable for downstream applications, such as PCR, following isolation from samples such as whole blood, buffy coat, or cultured cells.


Chapter 3 Assay Protocol16

Amount The iPLEX HS Colon and Lung Panels require at least 10 ng of DNA in a volume of 20 L for each PCR well. For example you can prepare a working dilution of 0.5 ng/μL and add 20 L of this dilution to each sample well. You may also use a smaller amount of a more highly concentrated working dilution plus molecular-grade water (e.g., 5 L of a DNA dilution of 2 ng/L plus 15 L of water).

IMPORTANT!It is strongly advised that the quality of the samples be checked using Agena Bioscience’s Exome QC Panel. This is to ensure that 3,000 - 5,000 amplifiable copies are used in the reaction, for optimal reaction conditions. Smaller or larger volumes of sample can be used by modifying the amount of water added.

Storage Purified genomic DNA can be stored at 4°C for up to 24 hours. To store DNA longer than 24 hours, we recommend storage at -20°C. Avoid repeated freezing and thawing of DNA.

For further detailed information on purification, storage, quantification, and analysis of genomic DNA, refer to the Qiagen website.

Preparing Sample DNA Plate

1. Aliquot 20 μL of each sample into a specific well of a new 96-well plate, as shown in Figure 3.1.

2. Seal the plate, centrifuge at 2,000 x g for one minute, and store at 4°C until ready for use.

3.2 Performing PCR Amplification

You will amplify genomic DNA from your samples using the supplied PCR primer in a final reaction volume of 50 μL.

IMPORTANT!Prepare the PCR cocktail and add it to the reaction plate in Lab Area 2. Thermocycle the PCR reaction plate in Lab Area 3. Make sure all reagents are thawed completely at room temperature and enzymes are kept on ice. Make sure reagents are homogenized before taking aliquots.

1. Prepare the PCR cocktail in a 1.5 mL microcentrifuge tube placed on ice by adding reagents in the order and quantities in which they are listed in Table 3.1.

Figure 3.1 Sample Plate


Chapter 3 Assay Protocol 17

Table 3.1 PCR Cocktail

*Includes a 25% overhang (for manual dispensing). Automated dispensing may require a higher overhang; contact Agena Bioscience for information on purchasing automation kits with higher fill volumes.

2. Vortex the tube for 3 seconds and briefly centrifuge.

3. Transfer 30 μL of PCR cocktail to wells A1-A12 of the sample plate, for a final PCR reaction volume of 50 μL.

4. Seal the plate, vortex for 5 seconds at high speed, then centrifuge at 3200 x g for 5 seconds.

5. Perform thermocycling using the following conditions:

3.3 Performing SAP Treatment

After amplification, any remaining free deoxynucleotides in the amplification reaction mixture must be dephosphorylated to prevent interference with the iPLEX HS reaction. Shrimp alkaline phosphatase (SAP) dephosphorylates unincorporated dNTPs and converts them to dNDPs, making them unavailable for the subsequent extension reactions. The SAP is then heat inactivated by incubation at 85°C for 5 minutes.

IMPORTANT!Prepare the SAP cocktail in Lab Area 2. Add the SAP cocktail to the reaction plate and thermocycle the plate in Lab Area 3. Make sure all reagents are thawed completely at room temperature and enzymes are kept on ice. Make sure all reagents are homogenized before taking aliquots.

.

1. Centrifuge the reaction plate at 3200 x g for 5 seconds.

2. Prepare the SAP cocktail in a 1.5 mL microcentrifuge tube on ice by adding reagents in the order and quantities in which they are listed in Table 3.2.

ReagentInitial

concentrationFinal

concentration1 well (μL)

12 samples (8 wells) (μL)*

Molecular grade water n/a n/a 8.4 126

10X PCR Buffer 10X 1X 5 75

MgCl2 25 mM 2 mM 4 60

dNTP Mix 25 mM 500 M 1 15

Global PCR Primer 0.5 M 0.1 M 10 150

PCR Enzyme 5 U/L 0.16 U/L 1.6 24

PCR Cocktail Volume 30 450

95°C 2 minutes 1 cycle

95°C 30 seconds

56°C 30 seconds 45 cycles

72°C 1 minute


15°C Hold



Table 3.2 SAP Cocktail



4. Dispense 20 μL of SAP cocktail into wells A1-A12 of the reaction plate.

5. Seal the reaction plate, vortex for 5 seconds at high speed, then centrifuge at 3200 x g for 5 seconds.

6. Perform thermocycling using the following conditions:

7. Aliquot 7 μL of the PCR/SAP product into each well of a new 96-well microtiter plate. Dispense from well A1 of the source plate to wells A1-H1 of the new plate; from well A2 of the source plate to wells A2-H2 of the new plate, etc.

Users running a 384 plate format should transfer 7 μL of analyte into appropriate wells of a new 384-well plate instead.

NOTEIf not proceeding directly to the next step, the reaction plate should be sealed and stored at -20°C. Do not store for more than 2 weeks.

ReagentInitial

ConcentrationFinal

concentration 1 well (μL)


Molecular grade water n/a n/a 15.3 229.5

SAP Buffer 10X 0.24X 1.7 25.5

SAP Enzyme 1.7 U/L 0.073 U/L 3.0 45.0

SAP Cocktail Final Volume 20 300

37°C 40 minutes


15°C Hold

Figure 3.2 New Extension Reaction Plate (numbers correspond to samples)



3.4 Performing the iPLEX HS Extension Reaction

NOTEIf the reaction plate has been stored frozen, let it thaw to room temperature before proceeding.

IMPORTANT!Prepare the iPLEX HS extension reaction cocktails in Lab Area 2. Add the extension reaction cocktails to the reaction plate and thermocycle the plate in Lab Area 3. Make sure all reagents are thawed completely at room temperature and enzymes are kept on ice. Make sure all reagents are homogenized before taking aliquots.

1. Centrifuge the reaction plate at 3200 x g for 5 seconds.

2. Prepare the iPLEX HS extension master mix in a 1.5 mL microcentrifuge tube placed on ice by adding reagents in the order and quantities in which they are listed in Table 3.3.

Table 3.3 iPLEX HS Extension Master Mix



4. Label eight 500 μL tubes from 1 to 8.

5. Prepare the extension reaction cocktails by adding master mix, extension primer, and termination mix to the eight tubes, as specified below:

a. Add 12.4 μL extension master mix to each tube.

b. Add 13.6 μL of the specific extension primer to the corresponding tube (add EXT W1 to tube 1, EXT W2 to tube 2, etc.).

c. Add 2.9 μL of the specific termination mix to the corresponding tube (add Termination Mix W1 to tube 1, Termination Mix W2 to tube 2, etc.).

Table 3.4 iPLEX HS Extension Reaction Cocktails


ReagentInitial

ConcentrationFinal


12 samples (96 wells)* (μL)


iPLEX Buffer Plus 10X 0.222X 0.20 25

iPLEX Pro Enzyme 225X 1X 0.04 5

Extension Master Mix Volume 0.86 107.4

Reagent 1 well (μL)12 samples (1

tube)* (μL)

Extension master mix 0.86 12.4

Extension primer 0.94 13.6

Termination mix 0.2 2.9

Extension Reaction Cocktail Total Volume 2.0 28.9



6. Vortex the tubes for 3 seconds and briefly centrifuge.

7. Dispense 2 μL of extension reaction cocktail into the appropriate wells of the reaction plate, as shown in Figure 3.3 (dispense from tube 1 to wells A1 - A12, from tube 2 to wells B1- B12, etc.).

8. Seal the plate with thermal sealing film, vortex for 5 seconds at high speed, then centrifuge at 3200 x g for 5 seconds.

9. Visually inspect the individual wells from the bottom of the reaction plates to confirm uniform and adequate solution is present in every well before continuing.

NOTEWorkflow dyes are compatible with these products; contact Customer Support (page 9) for information.

10. Thermocycle the reaction plate using the following reaction conditions:

NOTEIf not proceeding directly to the next step, the reaction plate should be sealed and stored at -20°C. Do not store for more than 2 weeks.

Figure 3.3 Reaction Plate(numbers correspond to a specific extension reaction cocktail)

95°C 30 seconds

95°C 5 seconds

52°C 5 seconds5 cycles 40

cycles80°C 5 seconds

72°C 3 minutes

15°C Hold



3.5 Next Steps: Processing Options

For 96- or 384-format experiments using the MassARRAY Nanodispenser and the MassARRAY Analyzer, proceed to Chapter 4.

For 96-format experiments using the MassARRAY System with Chip prep module, proceed to Chapter 5.


Chapter 4

MassARRAY Nanodispenser andMassARRAY Analyzer Processing

(96- or 384-format)

Desalting the iPLEX HS Extension Reaction Product (Dry Resin Method) . . . . . . . . . . . . 23Dispensing Samples to a SpectroCHIP Array (MassARRAY Nanodispenser) . . . . . . . . . 24Acquiring Data on the MassARRAY Analyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

This chapter gives instructions for desalting the extension products, dispensing onto a SpectroCHIP Array using the MassARRAY Nanodispenser, and acquiring data on the MassARRAY Analyzer for 96- or 384-format experiments only.

4.1 Desalting the iPLEX HS Extension Reaction Product (Dry Resin Method)

IMPORTANT!For 96-format experiments on a 96/384 MassARRAY System, analyte must be in a low profile plate for the dispensing step. If the assay protocol was performed in a non-low profile plate, it must be transfered to a low profile plate before proceeding. See Table 1.6 for recommended plate types.

IMPORTANT!Desalt the extension reaction in Lab Area 3. Wear gloves and safety glasses during the desalting procedure.

1. Centrifuge the reaction plate at 3200 x g for 1 minute.

2. Spoon 3 scoops of Clean Resin on a clean, dry dimple plate (96/15 mg plate or 384/6 mg plate as appropriate).

3. Spread out the Clean Resin on the dimple plate using the scraper, making sure the resin settles evenly into all wells.

4. Let resin plate dry for 10 minutes at room temperature.

5. While the resin plate is drying, add molecular grade water to each well of the reaction plate using a 12-channel multipipettor (41 L per well for 96-well plates; 16 L per well for 384-well plates).

6. Seal the plate and centrifuge at 3200 x g for 1 minute.

7. To add dried Clean Resin to each well:

a. Gently invert the sample plate on top of the dimple plate, making sure that the plate wells are aligned over the resin samples.

b. Keep the sample and dimple plates pressed together, and invert both plates so that the dimple plate is on top of the sample plate.

c. Gently tap the dimple plate and let the resin fall into the sample wells.

d. Remove dimple plate.


Chapter 4 MassARRAY Nanodispenser and MassARRAY Analyzer Processing (96- or 384-format)24

8. Seal the plate and rotate for at least 15 minutes. The rotator must rotate the microplate 360° around its long axis.

9. Centrifuge the plate at 3200 x g for 5 minutes to pellet the resin.

NOTEIf not proceeding directly to the next step, the reaction plate should be stored. Seal the reaction plate and store at -20°C.

4.2 Dispensing Samples to a SpectroCHIP Array (MassARRAY Nanodispenser)

NOTEIf the reaction plates have been stored frozen, let them thaw to room temperature and then rotate and centrifuge (at 3200 x g for 5 minutes) before transferring analyte to a SpectroCHIP Array.

The instructions presented here are for users who are familiar with operating the MassARRAY Nanodispenser. For more information, refer to the appropriate Nanodispenser user guide, available from Agena Bioscience Customer Support or from AgenaCx.com.

Before dispensing samples, confirm that the daily and weekly instrument maintenance procedures have been performed.

Preparing the Nanodispenser and Reaction Plate

1. Turn on the Nanodispenser and log on.

2. Check the supply and waste tanks, and the ultrasonic wash supply bottle.

3. Allow the calibrant to equilibrate to room temperature for 5 minutes (if it has been refrigerated) or 10 minutes (if it has been frozen). Fill the calibrant reservoir with 60 L of 3-Point calibrant.

4. Check to make sure that the resin levels and liquid are uniform across the microtiter plate wells. The liquid level must be at least 2.5 mm above the resin. See Appendix A for information on adjusting resin and liquid levels if necessary.

Selecting Nanodispenser Settings

1. Park the pin array assembly.

2. Load a method:

a. On the Main Menu screen, tap Transfer.


Chapter 4 MassARRAY Nanodispenser and MassARRAY Analyzer Processing (96- or 384-format) 25

b. On the Transfer screen, tap Methods, then on the Method screen tap Open.

c. Select the appropriate method file by tapping the file name.

d. Tap Open to load the file.

3. Tap the setup tab on the Method screen.

a. Specify the position on the SCOUT plate that will contain the SpectroCHIP Array by tapping the SpectroCHIP Array position that will be used.

b. Enable sample tracking if desired (requires a barcoded microtiter plate).

c. Enable volume check.

d. For instruments with v2.1 or higher software, enable auto tuning.

4. Tap the cleaning tab on the Method screen and confirm that the cleaning cycles are selected.

5. Tap the aspirate/dispense tab on the Method screen.

a. Under “dispense settings” enter a dispense speed of approximately 80-120 mm/sec, depending upon environmental conditions. The default target volume for dispensing iPLEX assays is 12 nL, but may range from 8 - 20 nL.

b. Under “calibrant” enter a dispense speed of 140 mm/sec.

c. Under “operation” choose analyte & calibrant.

6. Tap Back and apply or save settings.

Loading the Reaction Plate and SpectroCHIP Array

1. Place the 96- or 384-well reaction plate on the Nanodispenser processing deck, such that well A01 is to the lower left. (If you are using a non-skirted 96-well reaction plate, first place the reaction plate on the MTP plate base, then place on the deck.)

2. Remove the SCOUT plate from the processing deck and orient it so that the beveled corners are at the right. Using tweezers, insert a new SpectroCHIP Array into the SCOUT plate in the chip position you selected in Step 3 above. Orient the SpectroCHIP Arrays so that the Agena logo is at the bottom.

CAUTION!Do not touch the SpectroCHIP Array surface; handle only by the edges. Always wear gloves and use tweezers when handling SpectroCHIP Arrays.

3. Place the SCOUT plate back onto the processing deck.

4. Close the main door.

5. In the safety interlock is disengaged! dialog box, tap the HOME button.

Starting the Dispensing Run

For instruments with v2.1 or higher software:

1. On the Transfer screen, tap the Run button.

2. Tap OK in the rinse station preparation window that appears, and observe the rinse station operation.

3. If the rinse station operates properly, tap YES.The transfer will begin.



For instruments with v1.2 software:

1. On the Transfer screen, tap the step button to start the dispensing run.

2. When the instrument pauses, tap the volume button to check droplet volume. If necessary, adjust the dispense speed to get to a target volume of 12 nL per droplet.

3. Disable volume check, and then continue the run by tapping the run icon on the Transfer screen.

Removing the SpectroCHIP Array

1. Park the pin array assembly and, using tweezers, transfer the SpectroCHIP Array to the Chip holder for analysis in the MassARRAY Analyzer.

2. Remove the reaction plate, seal, and store at -20°C if you want to save the plate for future analysis.

3. If there is calibrant in the reservoir that will not be used within a few hours, remove the calibrant with a pipette and return it to the calibrant bottle for storage.

It is recommended that you process the SpectroCHIP Array immediately on the MassARRAY Analyzer. However, if you are not able to process the SpectroCHIP Array immediately, return it to the protective clamshell case and store in a dark desiccator with fresh desiccant for no longer than 2 hours.

4.3 Acquiring Data on the MassARRAY Analyzer

The instructions presented here for loading the SpectroCHIP Array into the MassARRAY Analyzer and acquiring data are for users who are familiar with operating the MassARRAY Analyzer. For more information, refer to the appropriate user’s guide (MassARRAY Analyzer 4 User Guide or MassARRAY Analyzer Compact User’s Guide) available from Agena Bioscience Customer Support or from AgenaCx.com.

Starting the MassARRAY Analyzer Software

To launch the MassARRAY Analyzer 4 application, double-click the Analyzer 4 instrument icon on the desktop.

To launch the MassARRAY Analyzer Compact application, double-click the Start RT

Processes icon on the desktop.

Loading the SpectroCHIP Array

NOTEAlways wear gloves and use the supplied tweezers when handling SpectroCHIP Arrays.

1. Move the Chip carrier out by pressing the manual control button on the front of the MassARRAY Analyzer, or by clicking Probe Sample In/Out or Target In/Out on the software toolbar. It takes approximately one minute for the Chip carrier to move to the load position.



CAUTION!When the Chip carrier is moving in or out of the MassARRAY Analyzer, do not start or quit data acquisition. Do not move the Chip carrier during spectra acquisition. Wait until acquisition is completed, or abort the run.

2. Open the sample chamber lid and take out the Chip holder. Remove any previously run SpectroCHIP Arrays from the Chip holder.

3. Place the SpectroCHIP Arrays to be processed into the Chip holder. Orient the SpectroCHIP Array in the Chip holder so that the Agena Bioscience logo is at the bottom. Make sure the lower left corner of the SpectroCHIP Array is flush against the Chip holder. SpectroCHIP Array 1 is seated on the left and SpectroCHIP Array 2 is seated on the right. If there is only one SpectroCHIP Array with samples, it is required that you place a second, dummy SpectroCHIP Array in the Chip holder. Also make sure that the dispensed analyte has dried and no liquid or dust is on the SpectroCHIP Array before loading.

4. Close the sample chamber lid and retract the Chip carrier by pressing the manual control button on the instrument, or selecting Target In/Out or Probe Sample In/Out in the software tool bar.

The Chip carrier moves to the “sample in” position. The MassARRAY Analyzer ready light illuminates when the required vacuum pressure (5 x 10-6 mbar) is reached. This takes approximately two minutes.

IMPORTANT!Only use the Chip holder that was supplied with your MassARRAY Analyzer. The Chip holder design is instrument-specific and Chip holders are not interchangeable between instruments.

Creating an Input File Using Chip Linker

An input file provides the information that SpectroACQUIRE needs to process a SpectroCHIP Array and save data to the MassARRAY database.

1. Double-click the Chip Linker icon on the desktop.

2. In the dialog box that appears, enter your user name, password, and server.



3. Click Connect. The Chip Linker window appears.

4. Select a plate in the Chip Linker directory tree.

5. Select the iPLEX Terminator Chemistry button.

6. Select Genotype+Area for the Process Method.

7. Select the dispenser method based on the SpectroCHIP Array format and dispenser mapping.

8. Enter an experiment name. When entering the information, use alphanumeric characters excluding special characters such as \/:*?<>|, and spaces.

9. For Chip Barcode, enter the barcode located on the SpectroCHIP Array.

10. Click Add. The input information appears in the Chip Linker table.

11. If a second SpectroCHIP Array will be processed, repeat step 4 to step 10 for the second SpectroCHIP Array.

12. Click Create to create an input .xml file. This file will be selected for use when you set up the automatic run.

13. Close the Chip Linker software.

Figure 4.1 Chip Linker Window



Setting Up the Automatic Run and Checking the Barcode Report

1. In SpectroACQUIRE, click the Automatic Run Setup tab (Figure 4.2).

2. Click the browse button next to Chip 1. In the dialog box that appears, select the input XML file created earlier (page 27) and click Open. Repeat for Chip 2 if necessary. (The chip number in the Automatic Run Setup tab corresponds to the SpectroCHIP Array position in the Chip holder. Chip 1 is seated on the left and Chip 2 is seated on the right.)

3. Select Tools > Load Parameters on the SpectroACQUIRE toolbar.

4. In the dialog box that appears, select the iPLEX.par file and click Open. This will load the parameter file. and populate the acquisition parameters fields. Make sure that the Filter Saturated Shots box is selected.

5. In the Geometry section, select your SpectroCHIP Array type from the Chip Type drop-down menu, and make sure that Use Calibration Wells and Auto Teach Geometry are selected.

6. It is recommended to select the Turn Off HV After Last Chip is Complete option in the Instrument section.

7. Click Barcode Report. In the dialog box that appears, each chip position should have a FOUND status. This means that the SpectroCHIP Array is properly associated with an experiment in the MassARRAY database.

8. Click CLOSE and correct any SpectroCHIP Array positions with an error status.

Starting the Automatic Run

1. Once the required vacuum pressure (5 x 10-6 mbars) is reached and the Ready light is illuminated, click the Automatic Run tab in SpectroACQUIRE.

Figure 4.2 SpectroACQUIRE Automatic Run Setup Tab



2. To start the run, click the button on the SpectroACQUIRE toolbar or select RunStart Auto Run on the menu bar.

The software:

• Confirms that the SpectroCHIP Array is associated with an experiment in the MassARRAY database.

• Confirms that the dispenser method selected in Chip Linker matches the plate size selected in Plate Editor.

• Checks the position of the SpectroCHIP Array in the Chip holder (i.e., performs auto teaching).

• Acquires calibration spectra if the Use Calibration Wells option has been chosen.

• Acquires spectra.

Unloading the SpectroCHIP Array

Do not unload SpectroCHIP Arrays while SpectroACQUIRE is acquiring spectra. Wait until the data acquisition is completed.

1. In the SpectroACQUIRE software, click the Automatic Run Setup tab.

2. Press the manual control button on the front of the MassARRAY Analyzer to extend the Chip carrier. It takes approximately one minute for the Chip carrier to move to the load position.

CAUTION!When the Chip carrier is moving in or out of the MassARRAY Analyzer, do not start or quit the SpectroACQUIRE software or start acquisition. Do not move the Chip carrier during spectra acquisition. Wait until acquisition is completed, or abort the run.

3. Open the sample chamber lid and take out the Chip holder. Remove the SpectroCHIP Arrays from the Chip holder. Replace Chip holder onto the Chip carrier. Press the manual control button on the front of the MassARRAY Analyzer to retract the Chip carrier.


Chapter 5

MassARRAY System with Chip prep moduleProcessing (96-format)

Preparing the Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Creating an Input File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Starting the Software and Checking Instrument Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Loading the Deck . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Setting Up the Run in the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Starting the Automatic Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Removing Plates and Calibrant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

This chapter gives instructions for desalting the extension products, dispensing onto a SpectroCHIP Array, and acquiring data using the MassARRAY System with Chip prep module for 96-format experiments only.

The instructions presented here are for users who are familiar with operating the MassARRAY System with Chip prep module. For more information, refer to the MassARRAY System with Chip prep module User Guide, available from Agena Bioscience Customer Support or from AgenaCx.com.

5.1 Preparing the Plate

1. Add 30 μL molecular grade water to each well of the reaction plate using a 12-channel multipipettor.

2. Centrifuge the plate at 3200 x g for 1 minute.

5.2 Creating an Input File

An input file must be created linking the virtual plate you created in MassARRAY Typer PlateEditor to a SpectroCHIP Array, and providing the information that the Analyzer needs to process the SpectroCHIP Array and save data to the MassARRAY database.

1. Double-click the Chip Linker icon on the desktop.

2. In the dialog box that appears, enter your user name, password, and server.

3. Click Connect. The Chip Linker window appears. See Figure 5.1.

4. Select a plate in the Chip Linker directory tree.

5. Select the iPLEX Terminator Chemistry button.

6. Select Genotype+Area for the Process Method.

7. Select Nanodispenser 96 to 96 as the dispenser method.

8. Enter an experiment name. When entering the information, use alphanumeric characters excluding special characters such as \/:*?<>|, and spaces.


Chapter 5 MassARRAY System with Chip prep module Processing (96-format)32

9. For Chip Barcode, enter the barcode located on the SpectroCHIP Array.

10. Click Add. The input information appears in the Chip Linker table.

11. If a second SpectroCHIP Array will be processed, repeat step 4 to step 10 for the second SpectroCHIP Array.

12. Click Create to create an input .xml file. This file will be selected for use when you set up the automatic run. Close the Chip Linker software.

5.3 Starting the Software and Checking Instrument Status

1. Double-click on the Start All icon to start MassARRAY Caller, Analyzer Control, Chip prep control (CPC), and SpectroACQUIRE.

2. The Status section in the Run Setup tab of SpectroACQUIRE indicates the readiness of the instrument. Waste Tank, System Fluid, and Resin buttons should be green/Okay. If any are red (Waste Tank Full, System Fluid Empty, Resin Low or Empty) perform the necessary maintenance.

Figure 5.1 Chip Linker Window


Chapter 5 MassARRAY System with Chip prep module Processing (96-format) 33

5.4 Loading the Deck

1. Click the Chip prep module Deck In/Out button at the top of the SpectroACQUIRE window to extend the deck. Remove any SpectroCHIP Arrays in the completed chips position.

2. Allow the calibrant to equilibrate to room temperature for 5 minutes (if it has been refrigerated) or 10 minutes (if it has been frozen). Pipette 75 μL of calibrant into the calibrant vial and place in the calibrant vial holder on the deck. One 75 μL vial of calibrant is enough for a day of operation (5 runs of 2 SpectroCHIP Arrays each).

3. Load the SpectroCHIP Arrays. SpectroCHIP Array 1 is seated on the left and SpectroCHIP Array 2 is seated on the right. If there is only one SpectroCHIP Array with samples, it is required that you place second, dummy SpectroCHIP Array in the chip holder.

IMPORTANT!Only use the chip holder provided with the MassARRAY System with Chip prep module, as other chip holders are not compatible with this instrument. The MassARRAY System with Chip prep module chip holder has slanted edges on its long sides (not straight edges).

a. Orient the chip holder so that the beveled corner is at the top right. Insert a new SpectroCHIP Array into the Chip holder in position 1; orient the SpectroCHIP Array such that the Agena logo and barcode are at the bottom. Make sure that the SpectroCHIP Array is flush against the chip holder.

CAUTION!Do not touch the SpectroCHIP Array surface; handle only by the edges. Always wear gloves and use tweezers when handling SpectroCHIP Arrays.

Figure 5.2 SpectroACQUIRE toolbar

Slanted edge Slanted edge



b. Place the full Chip holder in the Chip holder tray, new Chips position, on the deck.

4. Load microtiter plates. Place the first MTP on MTP holder 1 (the one on the left). This corresponds to Chip position 1. Orient the plate such that well A1 is in the lower left corner. If a second MTP is being processed, place it on MTP holder 2 (the one on the right). This corresponds to Chip position 2.

WARNING!WARNING! The Chip prep module is pre-configured to use the PCR plates specified in Table 1.6. If you wish to use different plates, contact Agena Bioscience Customer Support to update the instrument configuration.

5. Click the Chip prep module Deck In/Out button again to retract the deck.

5.5 Setting Up the Run in the Software

1. Select the Run Setup tab in SpectroACQUIRE.

Figure 5.3 Chip holder with SpectroCHIP Arrays

Chip position 1 Chip position 2

Beveled corner

Barcode and logo


Chapter 5 MassARRAY System with Chip prep module Processing (96-format) 35

Experiment Setup In the Experiment Setup section:

1. Under MTP 1, click on the browse button next to Experiment Name and select the XML input file created earlier (page 31).

2. In the Wells to Process field, select Automatic.

3. Repeat steps 1 and 2 for MTP 2 if running two plates. Otherwise, select None in the Experiment Name and Wells to Process fields for MTP 2.

4. In the Start Dispense Condition field, enter 500. This sets the initial volume and speed conditions for dispensing when using Autotune.

5. Check the Use Autotune box if autotune should be applied (recommended).

6. Enter 13 (μL) in the Resin Volume field.

7. Enter 7 (nL) in the Sample Volume field.

Chip prep module Setup

In the Chip prep module Setup section:

1. Check the Normal Operation box if doing a complete run with the chosen MTPs. Otherwise, check specific steps as needed.

2. Check MTP Barcodes Required if desired.If MTP Barcodes Required is checked and an MTP with a barcode is placed on the deck, then the barcode will be read and placed in an XML file labeled with the date and time, and the file will be placed in a folder at C:\ChipPrep\CPC\transferData. If MTP Barcodes Required is checked but the MTP placed on the deck does not have a barcode, an error message will be generated.

3. If the MTP and calibrant will not be removed from the instrument shortly after the run is completed, select the MTP Cool box and enter a temperature in the Setpoint box.

Figure 5.4 SpectroACQUIRE window: Run Setup tab



The MTP and calibrant will be kept at the desired temperature for up to 16 hours. 13°C is the recommended temperature.

4. Select the iPLEX from the Chemistry drop-down menu

Analyzer Setup The instrument comes pre-loaded with parameter files that specify the Analyzer Setup data acquisition parameters for specific chemistries. The iPLEX parameter file will automatically be loaded (because you selected iPLEX chemistry in your input file), and those values will auto-populate the Analyzer Setup section in the Run Setup tab. It is recommended that you do not change these Analyzer Setup values.

1. Be sure the Turn Off HV After Analysis, Filter Saturated Shots, and, if using calibrant, the Analyze Calibrant Pads boxes are checked.

2. Select SpectroCHIP-96 from the Chip Type drop-down menu.

Set Notification Options

If your instrument is networked, you may set the software to send you an e-mail notification when Chip prep module is finished dispensing from the plate to the Chip, and/or when the MassARRAY Analyzer has finished data acquisition.

To set up notification for a particular run, select the appropriate box(es) in the Run Setup tab. The software will also send notifications if any errors occur.

5.6 Starting the Automatic Run

In the toolbar at the top of the SpectroACQUIRE window, click the Start Chip prep module button to start the run. The MassARRAY System with Chip prep module will:

• Verify that the Chip prep module deck is properly loaded.

• Move any completed SpectroCHIP Arrays from the MassARRAY Analyzer to the completed Chips position on the deck.

• Perform all workflow steps checked in the Run Setup tab.

• Cool MTP and calibrant if selected.

Note that once the Chip prep module has transferred the SpectroCHIP Arrays to the MassARRAY Analyzer, you may load new SpectroCHIP Arrays and plates and begin the next run.

5.7 Removing Plates and Calibrant

Plates and calibrant should be removed and properly stored at the end of a run, unless the MTP Cool option was selected in the Run Setup tab.

1. Click the Chip prep module Deck In/Out button in the SpectroACQUIRE toolbar to move the deck out.

2. Remove the MTPs from the MTP holders and discard or store for future use if desired.

3. Remove the calibrant vial and store refrigerated or frozen for future use.


Chapter 6

Analyzing Data

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Generating Reports in TyperAnalyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Reading the iPLEX HS Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

6.1 Overview

Data analysis is performed using MassARRAY TyperAnalyzer. The instructions presented here are for users who are familiar with analyzing data with MassARRAY TyperAnalyzer. For comprehensive information on using the Typer application software, refer to the MassARRAY Typer 4 User Guide, which is available by contacting Agena Bioscience Customer Support or from AgenaCx.com.

Quality control A sample will be designated “failed” if any multiplex for that sample has a bad quality spectrum. Mutation calls are not made for failed samples.

Mutation call classification

Mutation status of the sample is determined based on the minor allele peak intensity and the overall mutation confidence. The software calls a mutation when the following two criteria are met:

• The minor peak intensity is above the minimum threshold (the default is a signal-to-noise ration [SNR] of 5). The user can adjust both the peak intensity type to be used (height, SNR, or area) and the minimum peak intensity value in the iPLEX HS Analysis Settings window (Figure 6.2).

• The minor allele frequency z-score exceeds the minimum z-score value (default is 3) designated in the iPLEX HS Analysis Settings window (Figure 6.2). The z-score is a “robust” z-score that represents the number of median absolute deviation (MAD) units that the minor allele’s frequency is away from the median of the baseline frequency for that allele.

The iPLEX HS Report will give both the mutation frequency and the adjusted mutation frequency, if available. The frequency is calculated by dividing the minor allele intensity by the WT allele intensity. That gives the observed experimentally-derived minor allele frequency.

Adjusted frequency is the observed frequency that is linearly transformed using the calibration coefficients for that assay. Calibration coefficients (slope and intercept) represent the linear correlation between the observed mutant allele frequency and the known percentage of the mutant allele for that assay. These data are obtained in-house by analyzing samples with known mutation frequencies. For the assays where the calibration coefficients are available the adjusted frequency will be different from the observed frequency. In these cases the adjusted frequency is a true mutation percentage.


Chapter 6 Analyzing Data38

IMPORTANT!For the iPLEX HS Lung Panel, see page 45 for instructions on eliminating possible false positives in the mutation report.

Baseline file It is recommended to use a baseline file of wild-type (non-mutant) control samples in order to reduce the likelihood of false negative calls in the event that the majority of samples being run have been previously ascertained as having mutations. There are two options for doing this; you will indicate which option you have selected in the iPLEX HS Analysis Settings window (Figure 6.2).

• Use the provided external assay baseline data file of 96 gDNA wild-type control samples (384-well plate). This option is recommended when analyzing fewer than 384-well datasets, or datasets in which the majority of the samples contain panel mutations. (Select Use External Assay Baseline Data in Figure 6.2.)

• Use wild-type control samples on the plate(s) you are running as the baseline data. In this case, you will specify each wild-type sample with a common name (e.g., NORM or WT). The software will use all samples with the common name (e.g., 1NORM, 2NORM, etc., or WT_1, WT_2, etc.) as the baseline data. (Specify the common name in the Text label for WT samples field in Figure 6.2.)

6.2 Generating Reports in TyperAnalyzer

1. Open TyperAnalyzer and in the Project Explorer pane double click on the SpectroCHIP Arrays of interest. The SpectroCHIP Arrays will be added to the Chip List.

2. Load the SpectroCHIP Arrays by checking the box next to the SpectroCHIP Array names in the Chip List.

3. Once the SpectroCHIP Arrays are loaded, select File > Reports > iPLEX HS Report in the TyperAnalyzer menu bar.

Figure 6.1 Generating Reports


Chapter 6 Analyzing Data 39

4. The Agena iPLEX HS Analysis Settings window will appear. If you do not wish to use the default settings, you may make changes here (see Table 6.1).

Table 6.1 iPLEX HS Analysis Settings

Figure 6.2 iPLEX HS Analysis Settings Window

Setting Default Description

Peak Intensity Type SNR May be Peak Height, SNR (signal-to-noise ratio), or Peak Area.

Peak Intensity Minimum

5 Minimum peak intensity in order for a peak to be analyzed.

Minimum Mutation Z-Score

3 Minimum mutation frequency z-score required for reporting the mutation.

Use External Assay Baseline Data

Selected This option is recommended when analyzing fewer than 384-well datasets, or datasets in which the majority of the samples contain panel mutations.

Text Label for WT Samples

Blank If you prefer to use wild-type control samples on the plate(s) you are running as the baseline data, designate each wild-type sample on the plate with a common name and enter that name here.



5. Click Run Analysis.The following window will appear while the iPLEX HS Report is being compiled.

When the report is complete, the results will be made available in two different formats (HTML and CSV) and stored in the Typer 4.0/bin/TyperReports/iPLEXHSReport folder in a folder named with the date and time. (The report desintation can be changed from the default location; contact Customer Support [page 9] for more information.) The HTML index file (the Home View) will automatically open in your browser.

NOTEIf the HTML reports do not open properly, make sure you are using a compatible browser, i.e., Internet Explorer (9, 10, or 10 Compatibility View), Firefox, or Chrome.

6.3 Reading the iPLEX HS Report

The CSV files contain all the data generated; the HTML files contain the data in a more readable format (Table 6.2). The HTML files are described in more detail below.

Table 6.2 iPLEX HS Report Files GeneratedFile type Folder or file name Description

HTML Home View Lists number of samples that passed and failed QC, number of samples with and without mutations, and lists all mutations tested for and the number of samples with those mutations.

Summary View Lists sample name, QC status, and mutations found.

Mutation View For a particular mutation, lists the samples which had that mutation and the mutation frequency.

Sample View For a particular sample, lists all detected and undetected mutations.

CSV iPLEXHSReport Lists each sample, its location, and the assay data for each (mutation frequency, adjusted frequency, z-score, whether simple or composite assays, QC status).

iPLEXHSBaseline Baseline file for the panel, including mutation intensities.

iPLEXHSBaselineProcessed Baseline file for the panel in which the mutation intensities have been converted to frequencies.

iPLEXHSLog Software data log for use by Agena Bioscience engineers during troubleshooting.

iPLEXHSRawData Raw data exported from MassARRAY Typer software.

iPLEXHSReportLong Lists the same data as the iPLEXHSReport, but in a long table format, where each assay for each sample is a row.



HTML Files Upon completion of report generation, the HTML-formatted Home View will automatically open in your browser window (Figure 6.3).

In each of the report views described below, you may:

• Change the z-score. Type the new z-score in the box at the top of the screen, and the view will be immediately updated to reflect the new parameter.

• If you change the z-score, you may want to export the new data to a CSV file. Click on the Export CSV button at the top of the screen to do so. This new file will reflect the results using the new z-score you have typed in the Change Zscore box.

• In views with columns, you may sort the data by column by clicking on the arrows at the top of the column.

Note for Internet Explorer users: Some additional steps are required to export data to a CSV file if you are viewing the HTML reports in Internet Explorer.

1. Click on the Export CSV button.

2. In the window that opens right-click and Select all.

3. Type Control-c to copy the entire window.

4. Open a new Excel document, click in cell A1, and type Control-v to paste the content into the new document.

5. Under the Data tab, select Text to columns.

6. Click Next in the window that appears, then in the following window, set the delimiters to Comma only, then click Finish.



Home View The Home View (Figure 6.3) lists the total number of samples tested on the selected SpectroCHIP Arrays, and the number of samples that passed QC, the number that failed QC, the number of samples with mutations, and the number of “normal” samples (samples without mutations; this includes NTC controls). Clicking on the sample number will take you to the Summary View (Figure 6.4) for that set of samples (e.g., all samples, those that failed QC, those with mutations, etc.)

Under Panel Content, each mutation tested for is listed, with the number of samples that have that mutation shown in parentheses. Mutations that were found will be shown in dark blue, and you can click on the mutation name to get to the Mutation View (Figure 6.6).

Figure 6.3 iPLEX HS Home View

Click on a number to go to the Summary View for this set of samples

Click on a mutation name to go to Mutation View



Summary View This view lists, for each sample, whether or not QC was passed, and the mutations found. Clicking on the sample name will take you to the Sample View (Figure 6.5).

Figure 6.4 iPLEX HS Summary View

Click a sample name to go to Sample View



Sample View The Sample View shows QC status and plate location for that particular sample, as well as a list of the mutations that were found, their frequency and adjusted frequency, and their Z-score.

Figure 6.5 iPLEX HS Sample View



Mutation View The Mutation View lists, for that particular mutation, all the samples in which that mutation was found and the mutation frequency and adjusted mutation frequency. Clicking on the sample name will take you to the Sample View (Figure 6.5).

Note for iPLEX HS Lung Panel

The iPLEX HS Lung Panel utilizes two assays to accurately identify complex mutations with multi-nucleotide base changes, large insertions, and deletions. A mutation is reported as detected when one or both assays identifies a mutant allele. This may lead to false positives being reported in some instances if only one assay has detected a mutant allele. When multiple mutations are reported that are not biologically possible (i.e., in the same location), the user is required to verify the spectra for both assays and eliminate false positives that may be listed in the report.

Probe nomenclatureThe assay or probe names provide details on the nucleotide position, base change, direction of the extension primer, and the amino acid change.

Example 1: EGFR_c2235GtoAf_Del19: c2235 is the nucleotide position, GtoA indicates G is the wildtype and A is the mutant allele, f indicates that the assay is in the forward or 3’ to 5’ direction for the EGFR exon 19 deletion.

Example 2: PIK3CA_c1633GtoAr_pE545K: c1633 is the nucleotide position, GtoA indicates G is the wildtype and A is the mutant allele, r indicates that this assay is the reverse direction, or 5’ to 3’, and the amino acid change is E545K.

Example AnalysisIn the example report for Sample13_1_LungHS (Figure 6.7), multiple EGFR#E746 mutations are listed in the Detected Mutation(s) section.

Figure 6.6 Mutation View

Click on a sample name to go to the Sample View.



To eliminate the false positives, use the following method:

1. Only EGFR#E746_A750delELREA has a high Zscore (28.92) and Frequency (0.32) as compared with the rest of the EGFR mutations, so start with this mutation. Use the assay table in Appendix D to identify the two probes required to detect this mutation (Figure 6.8 shows a selection from this table).The EGFR#E746_A750delELREA utilizes EGFR_c2235GtoAf_Del19 (Probe 1) and EGFR_c2249CtoAr_Del19 (Probe 2).

Figure 6.7 Example iPLEX HS Lung Panel Sample Report



2. Open Typer Analyzer and locate the sample and assay in the details pane. Verify that a peak is present for the mutant allele for both probes. Spectra A and B in Figure 6.9 show the presence of mutant allele peaks for probes 1 and 2 for the EGFR E746_A750delELREA deletion, indicating that it is a true mutation.

3. Repeat step 2 for all suspicious mutations listed in the report (EGRF#E746 mutations in this example).

a. For the E746_E749del mutation assay, a mutant allele peak is seen for probe 1 (Figure 6.10, spectra C), but the absence of a mutant peak in the spectra for probe 2 (spectra D) indicates that the mutation reported is not true, and is a false positive.

Figure 6.9 Sample 13_1, EGFR#E746_A750delELREA mutation

Figure 6.8 Selection from iPLEX HS Lung Panel Assay Table (Appendix D)

A. Probe 1: EGFR_c2235GtoAf_Del19

Mut A peak present

B. Probe 2: EGFR_c2249CtoAr_Del19

Mut A peak present



Figure 6.10 Sample 13_1, EGRF#E746_E749del mutation

b. Probe 1 is common to EGFR E746_E749del, E746_T751>I, E746_A750>IP, E746_S752>I, E746_T751>IP, and E746_A750delELREA mutations, and has a true mutant allele peak. However, only probe 2 assay EGFR_c2249CtoAr_Del19 has a true mutation peak, making the E746_A750delELREA a true mutation.

c. In this example, E746_T751>IP may also be a true mutation, give that probe 1 shows the presence of the mutant allele, and this mutation does not have a second probe. However, E746_T751>IP is a complex inframe deletion with a much lower frequency of occurrence as compared with the 15bp E746_A750delELREA deletion and is not true.

4. For suspicious mutations which may be caused by two different changes, the user must manually verify the spectra for all possible combinations to confirm the mutation, because the iPLEX HS mutation report does not identify which of the two changes was detected, or which probes were used to detect the mutation. In this example, EGFR#E746_A750delELREA may be caused by two different deletions: a 15bp deletion between c.2235 and c.2249 or between c.2236 and c.2250 (see lines 2 and 7 in Figure 6.8).

C. Probe 1: EGFR_c2235GtoAf_Del19

Mut A peak present

D. Probe 2: EGFR_c2245GtoAr_Del19

Mut A peak not present


Appendix A

Troubleshooting

The following tables list some potential problems and solutions that may help you troubleshoot your experiments. More information may be found in the following user guides, all available at AgenaCx.com or by contacting Agena Bioscience Customer Support (page 9).

• MassARRAY Typer 4 User Guide

• MassARRAY Nanodispenser RS1000 User Guide

• MassARRAY Analyzer 4 User Guide

• MassARRAY Analyzer Compact User Guide

• MassARRAY System with Chip prep module User Guide

Troubleshooting the iPLEX HS Report

Table A.1 Troubleshooting (iPLEX HS Report)

Troubleshooting MassARRAY Nanodispenser and MassARRAY Analyzer

Table A.2 Troubleshooting (MassARRAY Nanodispenser and MassARRAY Analyzer)

Problem Possible Reason Possible Solution

Error when generating reports.

R is not properly installed. Re-install R. See MassARRAY Typer Release Notes for instructions.

HTML reports are not displaying correctly.

Not using compatible browser. See page 8 for a list of compatible browsers.


Too much resin in a well. Uneven analyte or resin transfer.

Carefully pipette out some resin, removing only what is necessary. Centrifuge the plate for 3 minutes at 1600 x g. See MassARRAY Nanodispenser RS1000 User Guide for more information.

Liquid level in a well is less than 2.5 mm above the resin.

Uneven analyte or resin transfer.

Bring up the liquid level by adding molecular grade water with a pipette. See MassARRAY Nanodispenser RS1000 User Guide for more information.

Some wells have lower liquid level than others.

Uneven analyte or resin transfer.

Bring up the liquid level in low wells by adding molecular grade water with a pipette. See MassARRAY Nanodispenser RS1000 User Guide for more information.

Analyte volume dispensed onto the SpectroCHIP Array is too low or highly variable.

Low humidity or sample-to-sample variability.

At the desalting step, use a 0.001% Tween and molecular grade water solution in place of the regular water addition.


Appendix A Troubleshooting50

The Analyzer 4 takes longer than three minutes to reach the required vacuum pressure (5 x 10-6 mbars)

Sample chamber does not properly seal.

Wipe the mating surface of the sample chamber lid with a lint-free tissue that is moistened with 50% isopropyl alcohol or ethanol. Leave the sample chamber lid open until the surface is dry.

Remove the sample chamber O-ring. Wipe the O-ring with a lint-free tissue and replace it in the O-ring groove after cleaning out the groove with a lint-free tissue moistened with alcohol as above.

NOTEDo not allow the O-ring to come in contact with alcohol. Allow the groove to dry before reinserting the O-ring.

False positives observed. Clean Resin is too dry (dry resin method) or was not added to plate.

Ensure Clean Resin was added. Do not dry resin more than 5 - 10 minutes. Rotate the plate for an additional 30 - 45 minutes.

Mutation frequency cutoff is too low.

Adjust the mutation frequency z-score (default is 3).

Peak intensities are too low.

Laser energy is too low. Before running the SpectroCHIP Array on the MassARRAY Analyzer, fire on one of the calibrant pads and check that the calibrant peaks are 100 +/- 25%. Contact Agena Bioscience Customer Support if low.

Correct parameter file not loaded.

Check that the acquisition parameters are as shown in Figure 4.2.

SpectroCHIP Arrays are over- or under-dispensed.

Perform a volume check to make sure correct volumes are dispensed on the SpectroCHIP Array. See the MassARRAY Nanodispenser RS1000 User Guide for instructions.

Peak shape is too wide. MassARRAY Analyzer is not properly tuned.

Contact Agena Bioscience Customer Support.

Non template control wells have calls.

PCR contamination. Ensure pre-PCR preparations are performed in a UV-sterilized chamber.

Self-extension of Extend primers.

Repeat the experiment or redesign the Extend primers.

Peaks are not aligned to the expected calls.

Calibration may not be enabled in the SpectroACQUIRE software.

Ensure calibration is enabled. See page 29.

MassARRAY Analyzer is not properly tuned.




Appendix A Troubleshooting 51

No calls. Unextended peaks present due to iPLEX Reagent Set enzyme, terminator mix, or buffer missing from the master mix.

Repeat the experiment.

Unextended peaks present. Ensure mastermix and thermocycling protocols were followed and PCR inhibitor (e.g., EDTA) is not present in samples.

No unextended peaks due to Extend primer mix missing from the master mix.


Some wells have bad or failed spectra.

Make sure the Nanodispenser is properly calibrated to the plate; this will ensure that the pins access the analyte. See the MassARRAY Nanodispenser v2.1 User Guide for more information.

Peaks are present in the spectra but not at the expected masses, due to the wrong assay definition being applied in PlateEditor.

Recall the plate in PlateEditor and apply the correct assay definition. See MassARRAY Typer 4 User Guide for more information. Repeat the experiment.

Peaks are present in the spectra but not at the expected masses, due to the wrong Extension mix being added.


Peaks are present in the spectra but not at the expected masses, due to the MassARRAY Analyzer needing tuning.


SpectroCHIP Array improperly linked to a plate/dispenser method.

Verify that the SpectroCHIP Array is properly linked to an experiment in the database.




Troubleshooting MassARRAY System with Chip prep module

Table A.3 Troubleshooting (MassARRAY System with Chip prep module)Problem Recommended Action

Problems with plate and SpectroCHIP Array processing

The camera failed and the software was unable to connect (Error code 3).


Chip or Chip barcode is not present (Error codes 12, 13, or 46).

Verify that the SpectroCHIP Array is present, installed correctly in the Chip holder, and not dirty: 1. Click the Deck In/Out button in SpectroACQUIRE.2. Remove the SpectroCHIP Array (using tweezers).

3. Use clean dry air to blow gently over the SpectroCHIP Array.

4. Replace the SpectroCHIP Array back in the Chip holder on the deck and retract the deck. See Figure 5.3 for proper positioning of SpectroCHIP Array.

The wash station is full (error code 54).


Unable to locate a pad during autoteaching (error code 63).

Verify that the SpectroCHIP Array is present, installed correctly in the Chip holder, and not dirty: 1. Click the Deck In/Out button in SpectroACQUIRE.2. Remove the SpectroCHIP Array (using tweezers).

3. Use clean dry air to blow gently over the SpectroCHIP Array.

Replace the SpectroCHIP Array back in the Chip holder on the deck and retract the deck. See Figure 5.3 for proper positioning of SpectroCHIP Array.

Resin or resin tray problem (error codes 4 or 55).

In the Run Setup tab of SpectroACQUIRE, click on the Refill/Maintain Resin Tray button. Verify that the resin tray in present, properly installed, and contains resin.

Unable to find the MassARRAY Analyzer lid (error code 73).


Problems with mass spectrometry results

Peak intensities are too low.


Peak shape is too wide. The MassARRAY Analyzer may need tuning. Contact Agena Bioscience Customer Support.

Non template control wells have calls.

Ensure pre-PCR preparations are performed in a UV-sterilized chamber. If due to self-extension of Extend primers, repeat the experiment, or redesign the Extend primers.

Peaks are not aligned to the expected calls.

Ensure that calibrant was dispensed to the SpectroCHIP Array, and that calibration is enabled in the SpectroAQUIRE software (see page 35).

If that does not correct the problem, the MassARRAY Analyzer may need tuning. Contact Agena Bioscience Customer Support.

No calls. Verify that the SpectroCHIP Array is properly linked to an experiment in the database (see page 31).

If that does not correct the problem, and peaks are present in the spectra but not at the expected masses, the MassARRAY Analyzer may need tuning. Contact Agena Bioscience Customer Support.


Appendix A Troubleshooting 53

Table A.4 Chip prep module Front Panel Indicator Lights

Blue (Power)Green (Working)

Red (Error) Meaning

Off Off Off Instrument is not powered on.

Off Off Blinking Software or hardware failure; contact Agena Bioscience Customer Support.

On Off Off The Chip prep module may not be powered on. Make sure that the power button on the back of the Chip prep module instrument is on. If that does not resolve the problem, contact Agena Bioscience Customer Support.

On Off On Instrument is not ready to run. Verify that waste tank is not full, system fluid tank is not empty, and resin tray is full. If still getting error, contact Agena Bioscience Customer Support.

On Off Blinking steadily SpectroACQUIRE software may not be running. Make sure that the software is running. If that does not resolve the problem, contact Agena Bioscience Customer Support.

On Off Blinking (two flashes separated by a pause)

Instrument is paused because one of the doors in open. Make sure all doors are closed.

On On Blinking (three flashes separated by a pause)

Resin tray may not be properly covered. In the Run Setup tab of SpectroACQUIRE, click on the Refill/Maintain Resin Tray button. Verify that the resin tray is present, properly installed, and contains resin.

If that does not resolve the problem, contact Agena Bioscience Customer Support.

On On Off Instrument is idle but ready to run.

On Blinking Off A run or autowash cycle is in progress.


Appendix B

Quick Reference Guide: Reaction Conditions

B.1 PCR Reaction Conditions

Prepare the PCR cocktail for as many reactions as you need in a 1.5 mL microcentrifuge tube placed on ice by adding reagents in the order in which they are listed in Table B.1. Add DNA separately.

Table B.1 PCR Reaction Conditions


Table B.2 PCR Thermocycling Conditions

B.2 SAP Reaction Conditions

Prepare the SAP cocktail in a 1.5 ml microcentrifuge tube on ice by adding reagents in the order in which they are listed in Table B.3.

Table B.3 SAP Cocktail


ReagentInitial

concentrationFinal



Molecular grade water n/a n/a 8.4 126

10X PCR Buffer 10X 1X 5 75

MgCl2 25 mM 2 mM 4 60

dNTP Mix 25 mM 500 M 1 15

Global PCR Primer 0.5 M 0.1 M 10 150

PCR Enzyme 5 U/L 0.16 U/L 1.6 24

PCR Cocktail Volume 30 450

DNA (add separately) 20

PCR Reaction Final Volume 50


95°C 30 seconds

56°C 30 seconds 45 cycles

72°C 1 minute


15°C Hold

ReagentInitial

ConcentrationFinal

concentration 1 well (μL)



SAP Buffer 10X 0.24X 1.7 25.5

SAP Enzyme 1.7 U/L 0.073 U/L 3.0 45.0

SAP Cocktail FInal Volume 20 300


Appendix B Quick Reference Guide: Reaction Conditions56

Table B.4 SAP Thermocycling Conditions

B.3 iPLEX HS Extension Reaction Conditions

Prepare the iPLEX HS extension master mix in a 1.5 ml microcentrifuge tube placed on ice by adding reagents in the order in which they are listed in Table B.5. Add each extension primer and termination mix separately.

Table B.5 iPLEX HS Extension Master Mix


Table B.6 Extension Primer and Termination Mix Addition


Table B.7 Extension Reaction Thermocyling Conditions

37°C 40 minutes


15°C Hold

ReagentInitial

ConcentrationFinal


12 samples )96 wells)* (μL)


iPLEX Buffer Plus 10X 0.222X 0.20 25

iPLEX Pro Enzyme 225X 1X 0.04 5

Extension Master Mix Volume 0.86 107.4

Reagent 1 well (μL)12 samples (1

tube)* (μL)

Extension master mix 0.86 12.4

Extension primer 0.94 13.6

Termination mix 0.2 2.9

Extension Reaction Cocktail Total Volume 2.0 28.9

95°C 30 seconds

95°C 5 seconds

52°C 5 seconds5 cycles 40

cycles80°C 5 seconds

72°C 3 minutes

15°C Hold


Appendix C

Additional Peaks

Known Peaks Commonly observed adducts are sodium (22 Da), potassium (38 Da), manganese (55 Da) and matrix (189 Da). The sodium and potassium adducts are the most problematic because they are very close to peak separations between C to A mutations (24 Da) and G to T mutations (40 Da). Several steps have been taken to reduce the presence of adducts and to minimize their impact on data analysis, including applying an adduct correction algorithm as part of the software. Allele frequencies are calculated after automatic adduct correction.

Unknown Peaks Several peaks that are called as unknown by the MassARRAY Typer software may be the result of depurination or depyrimidation. Most of these peaks do not interfere with calls.

Depurination/depyrimidation peaks can be the result of extend primer degradation from freeze-thaw cycles, or from the MassARRAY Analyzer laser being set too high. Table C.1 shows the expected peaks from each depurination or depyrimidation event, and Figure C.1 shows a small peak at -150 from the main peak, corresponding to a guanidine depurination.

Table C.1 Expected Peaks from Depurination/DepyrimidationEvent

Depurination A -134 Da

G -150 Da

Depyrimidation C -110 Da

T -125 Da

Figure C.1 Example Unknown Peak (boxed in red) Showing a G Depurination


Appendix C Additional Peaks58



Appendix D

iPLEX HS Lung Panel Assays

Table D.1 iPLEX HS Lung Panel Assays


Appendix D iPLEX HS Lung Panel Assays60


Appendix D iPLEX HS Lung Panel Assays 61


Appendix D iPLEX HS Lung Panel Assays62


Appendix E

Licensing

Warranty. Standard Agena warranties shall apply as set forth in the Agena MassARRAY® System Warranty documentation provided with each System. EXCEPT AS SET FORTH IN THIS SECTION 5, AGENA MAKES NO REPRESENTATIONS, WARRANTIES, GUARANTEES, OR CLAIMS, OF ANY KIND OR NATURE, AS TO THE ACCURACY OF, RELIABILITY, UTILITY, PERFORMANCE, EFFECTIVENESS, OR OTHERWISE, OF THE SYSTEM PROVIDED HEREIN OR THE RESULTS OBTAINED THEREFROM, NOR DOES AGENA ASSUME ANY RESPONSIBILITY FOR THE RESULTS, QUALITY OF RESULTS, OR LACK OF RESULTS THE SYSTEM AND REAGENTS ARE PROVIDED "AS IS" AND WITHOUT WARRANTY OF ANY KIND, WRITTEN OR ORAL, EXPRESS OR IMPLIED, DIRECT, INDIRECT, BY ESTOPPEL, OR OTHERWISE, AND SPECIFICALLY EXCLUDING ANY IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE, NON-INFRINGEMENT OF ANY THIRD PARTY PATENT OR OTHER INTELLECTUAL PROPERTY RIGHTS, AND ANY OTHER WARRANTIES PROVIDED FOR BY THE UNIFORM COMMERCIAL CODE, USAGE IN THE INDUSTRY, OR COURSE OF DEALING BETWEEN THE PARTIES.

License. Agena’s patented nucleic acid analysis by mass spectrometry methods and products are protected under United States patent rights and patents pending, and foreign counterparts. With the purchase of Agena SpectroCHIP® arrays and reagent kit products Customer is granted a limited right to practice Agena’s patented methods and use its products. The licensed rights are not transferable, are for the benefit of Customer only, and expire with the consumption of the SpectroCHIP® arrays purchased. Transfer or resale of products and their components, purchased from Agena, is prohibited. The license rights granted are limited to one-time use only for each element per SpectroCHIP® array purchased. For example, Agena’s SpectroCHIP® 384 Array is provided with 384 elements and each element may be used only once. Re-manufacture or re-use of Agena’s SpectroCHIP® arrays and/or elements in conjunction with performing Agena’s patented nucleic acid analysis methods, is prohibited. Reverse engineering Agena products is prohibited. Except as expressly licensed with the purchase of chips and reagent kit products as set forth herein, Agena reserves all rights and no additional license rights are granted or implied.

Software License. For purposes of this Agreement, “Software” means computer software or programs supplied by Agena to Customer including but not limited to such software that is embedded in or forms an integral part of Agena’s hardware products in addition to separately provided software for application specific purposes. For purposes of this Agreement, Software does not include Oracle Corporation software products provided by Agena as such Oracle products are separately governed by the attached Oracle End User License Terms.

(a) Agena grants Customer a non-exclusive and non-transferable license to use the Software for processing data exclusively in conjunction with specially designated SpectroCHIP® arrays and reagents or reagent kit products purchased from Agena. Customer shall not permit another party to use the Software and Customer shall effect and maintain adequate security measures to safeguard the Software from access or use by unauthorized persons. Customer shall not transfer, rent, lease, sublicense, loan, copy, modify, adapt, merge, translate, reverse engineer, decompile, or disassemble the Software or create derivative works based on the whole or any part of the Software.


Appendix E Licensing64

(b) The Software license shall not be deemed to extend to any of Agena’s intellectual property rights, including rights in source code. No copies may be made of the Software without the prior written consent of Agena, except that Customer may make a single back up or archival copy. The Software license shall apply to any copy as it applies to the Software.

(c) With specific reference to application Software and including any associated report tools, Customer is provided a royalty-free, non-exclusive and non-transferable license to use the Software subject to the terms and conditions in subsections (a) and (b) above, solely upon the condition that such Software is used exclusively in conjunction with specially designated SpectroCHIP® arrays and reagents or reagent kit products purchased from Agena.

(d) All Software licenses shall terminate automatically and immediately if Customer fails to abide by any of the terms and conditions of this Agreement. Except as expressly licensed herein, Agena reserves all rights. Other than as expressly set forth herein, no license rights are granted or implied.

No Governmental Approval. Customer is hereby put on notice that all of Agena’s products and Software are Research Use Only and have not been subjected to regulatory review or cleared or approved by the United States Food and Drug Administration, or any other United States governmental agency or entity, or by any similar governmental agency outside the United States, and are not CLIA (or the regional equivalent thereof) registered or licensed or otherwise registered, licensed, or approved under any statute, rule, law, or regulation for any purpose. Customer bears sole responsibility and liability for validating all products purchased for Customer’s intended use.

Indemnity. Customer will indemnify and hold harmless Agena Bioscience, Inc., its subsidiaries, and all officers, directors, employees and agents of the foregoing, (the “Agena Indemnified Parties”) from and against any and all losses, damages, liabilities, expenses and costs, including responsible legal expense and attorneys’ fees (“Losses”), to which a Agena Indemnified Party may become subject as a result of any claim, demand, action or other proceeding by any third party to the extent such Losses arise from acts or omissions by Customer resulting in (a) personal injury or harm from the use, sale or other disposition of any product or service or information provided to a third party by Customer and/or its contractual partners, customers, or collaborators; or (b) a claim that a Customer product, assay, test analysis, method, or procedure infringes any patent, trade secret or other intellectual property rights of any third party, unless such claim is solely based on Customer’s non-assay-specific use of Agena’s products according to the authorized and intended use for such product and in compliance with user operating manuals and package inserts provided with such products. The foregoing shall not apply where such Losses are caused by Agena’s gross negligence or willful misconduct. In the event an Agena Indemnified Party seeks indemnification under this provision, Agena will notify Customer in writing of a claim as soon as reasonably practicable after it receives notice of the claim, will permit Customer to assume direction and control of the defense of the claim (including the right to settle the claim) and will cooperate as requested (at the expense of Customer) in the defense of the claim. This Section will survive the expiration or termination of this Agreement.


Appendix E Licensing 65

Oracle end user license terms

ORACLE CORPORATION ("ORACLE") SOFTWARE PRODUCTS

(a)The Customer shall limit its use of Oracle's products to the scope of the application package and to its business operations;

(b)The Customer shall not transfer Oracle's products except for temporary transfer in the event of computer malfunction;

(c)The Customer shall not assign Oracle's products or any interest in Oracle's products. If a security interest is granted in Oracle's products, the secured party has no right to use or transfer Oracle's products;

(d)The Customer shall not operate a timeshare, hosting, subscription service, or rental use of Oracle's products;

(e)Title to Oracle's products remains with Oracle and shall not pass to the Customer or to any other party;

(f)The Customer shall not reverse engineer, disassemble or decompile Oracle's products unless required for interoperability and then only to the extent so required for such interoperability;

(g)The Customer shall not duplicate Oracle's products except for a sufficient number of copies for the Customer's licensed use and a single back up or archival copy;

(h)Oracle shall not be liable for any damages whether indirect, incidental, special, punitive, or consequential arising from the use of its products;

(i)Where the license which is granted by Agena in respect of Oracle products expires or terminates and is not renewed, the Customer shall discontinue use and destroy or return all copies of Oracle's products and documentation to Agena;

( j)The Customer shall not cause to be publicized any results of benchmark tests run on Oracle's products;

(k)The Customer shall comply fully with all relevant export laws and regulations of the United States and other applicable export and import laws to assure that neither the Oracle products themselves nor any direct products thereof are exported, directly or indirectly in violation of applicable laws;

(l)Oracle is not required to perform any obligations other than to the extent agreed by Agena and Oracle;

(m)Agena has the right to audit Customer's use of Oracle's products and report such use to Oracle, or to assign such right to another person;

(n)Oracle is a third party beneficiary of these Customer license terms;

(o)The Uniform Computer Information Transactions Act shall not apply; and

(p)To the extent that Oracle source code is included with any application package, such source code is similarly governed by the terms above.

[August 2015]


Appendix E Licensing66



Appendix F

Warranty

RESEARCH USE ONLY Customer is hereby put on notice that AGENA BIOSCIENCE (“AGENA”) products (including the MassARRAY System, and all hardware, software, and consumables) are Research Use Only and have not been subjected to regulatory review or cleared or approved by the United States Food and Drug Administration, by any other United States governmental agency or entity, or by any equivalent or similar governmental agency or entity outside the United States, and are not CLIA (or the regional equivalent thereof) registered or licensed or otherwise registered, licensed, or approved under any statute, rule, law, or regulation, for any purpose, whether commercial, diagnostic, medical, or otherwise. Customer bears sole responsibility and liability for validating all products purchased by Customer for Customer’s intended use. Customer assumes the entire risk as to the results and performance of AGENA’s products for Customer’s specific applications and use. “CLIA” means the Clinical Laboratory Improvement Amendments, 1988. Customer assumes full responsibility for the results and use of the results obtained from the use of AGENA’s products and related documentation. AGENA’s products (including the MassARRAY System, and all hardware, software, and consumables) are for research use only and are not to be used for diagnostic or medical purposes.

LIMITED WARRANTY Warranty. This limited warranty extends only to Customer as original purchaser unless otherwise agreed upon in writing by AGENA. AGENA warrants that the MassARRAY System (meaning hardware and installed software but excluding consumables such as kits, chips, and reagents) will be free from defects in materials and workmanship and will conform to AGENA’s specifications in effect as of the date of delivery, and perform accordingly, until the earlier of (i) one (1) year from the date of installation; or (ii) sixteen (16) months from the date of shipment, (the “Warranty Period”) and only so long as the MassARRAY System remains unchanged and in the original condition supplied by AGENA. AGENA warrants that MassARRAY consumables (including but not limited to kits, chips, and reagents) will be free from defects in materials and workmanship and will conform to AGENA’s specifications in effect as of the date of delivery, and perform accordingly from the date of delivery up to the expiration date specified on the product packaging (the “Warranty Period”), provided that such consumables have been stored, handled, and used according to AGENA specifications, and otherwise remain unchanged and in the original condition supplied by AGENA.

Warranty Exclusions. The foregoing warranty does not include, and Customer is responsible for (i) periodic maintenance or calibration recommended for certain products; (ii) except for lasers, the replacement of hardware components such as but not limited to, dispense pins, liquid handler tips, and clean resin tools; (iii) maintaining a procedure to back-up any and all data generated by the MassARRAY System and reconstructing any lost or altered files, data, or programs; (iv) maintaining all computer networking as it relates to the integration of any components of the MassARRAY System outside of the System and within Customer’s networks; (v) proper disposal of waste products (e.g., oil, filter elements) that result from service work performed under the warranty. Further, the foregoing warranty shall not apply to an AGENA product that (i) has been subjected to improper or inadequate maintenance or calibration by Customer; (ii) has been used with third party hardware, software, interfacing, or other products or components provided by parties other than AGENA and not approved in writing by AGENA; (iii) has been used to process samples loaded on re-used and/or refurbished AGENA chip products (including

SpectroCHIP® Arrays); (iv) has been repaired, modified, altered, assembled or


Appendix F Warranty68

disassembled, or moved from its original installation location, by persons other than AGENA or an AGENA authorized service provider; (v) has not been maintained, calibrated, operated or installed in accordance with AGENA’s written specifications including operator failure to perform standard operating procedures and routine maintenance as prescribed in the user guides; (vi) has been subjected to abuse, misuse, negligence, neglect, accident, or improper storage, installation, operation, or handling contrary to AGENA’s written specifications or any instructions provided by AGENA; (vii) has failed or been damaged due to environmental conditions at the site of installation including exposure to Bio- Safety Level 3 or 4 (as defined by the United States Occupational Health and Safety Administration) agents; exposure to radioactivity; or the use of or invasion by malicious software programs such as viruses; or (viii) has failed or been damaged due to causes beyond its reasonable control, including, but not limited to, acts of civil or military authority, riots, wars, terrorism, fires, floods, earthquakes, lightning, storms, and other acts of God.

Remedy. If during the Warranty Period AGENA is provided with prompt written notice by Customer of any product failure along with a detailed written description of the failure, then upon AGENA’s inspection and testing as may be necessary to confirm the failure and upon its determination that none of the warranty exclusions above are applicable, AGENA shall at its sole discretion and option, as Customer’s sole remedy and AGENA’s sole obligation under the foregoing warranty: (1) replace the products in whole or components thereof; or (2) repair (and recalibrate as necessitated by repair) the products. Replacement and repaired products and components may include functionally equivalent, reconditioned, refurbished, or new parts. For consumables, provided that AGENA has confirmed the failure and determined that none of the warranty exclusions above are applicable, AGENA reserves the right at its sole discretion to either replace the consumables or to provide Customer with credit for the purchase price paid only, prorated for the unusable portion of the consumables, to be used to offset future purchases. No refunds will be made. Depending upon the type of failure as determined by AGENA, if AGENA needs access to Customer’s facility, Customer shall provide AGENA with access to its facility and information deemed necessary to facilitate a repair, or if directed by AGENA, Customer shall return the products, in whole or in part, at AGENA’s expense per AGENA’s specific instructions provided at the time, or if no specific instructions are provided at the time, in accordance with AGENA’s then current written return procedures (which may be amended from time to time). Prior to any service visit, Customer must notify AGENA in writing of any decontamination of the MassARRAY System or any product that has been exposed to toxic or dangerous substances. No products or components may be returned to AGENA under any circumstances without AGENA's prior authorization. During service performed at Customer’s facility, Customer must have a Customer representative on-site at all times. Any product or component that has been repaired or replaced under the warranty shall have warranty coverage for the remainder of the original Warranty Period.

EXCEPT FOR THE EXPRESS LIMITED WARRANTY SET FORTH HEREIN, AGENA MAKES NO OTHER WARRANTIES, ORAL OR WRITTEN, EXPRESS, IMPLIED, OR BY STATUTE, WITH RESPECT TO THE MASSARRAY SYSTEM, OR ANY HARDWARE, SOFTWARE, CONSUMABLES, COMPONENTS, AND DOCUMENTATION REGARDING ANY OF THE FOREGOING, INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, NONINFRINGEMENT OF PATENT RIGHTS OR VIOLATION OF INTELLECTUAL PROPERTY RIGHTS OF ANY KIND, OR ARISING FROM COURSE OF DEALING, PERFORMANCE, USAGE OR TRADE. FOR THE AVOIDANCE OF DOUBT, AGENA DOES NOT WARRANT, GUARANTEE, OR MAKE ANY REPRESENTATIONS REGARDING THE USE OR THE RESULTS OF THE USE, OF THE


Appendix F Warranty 69

MASSARRAY SYSTEM OR ANY HARDWARE, SOFTWARE, CONSUMABLES, COMPONENTS, AND DOCUMENTATION IN TERMS OF CORRECTNESS, ACCURACY, RELIABILITY, OR OTHERWISE.

LIMITED LIABILITY IN NO EVENT SHALL AGENA OR ITS SUPPLIERS BE LIABLE TO CUSTOMER OR ANY THIRD PARTY FOR LOST PROFITS, DATA OR BUSINESS, THE COSTS OF PROCUREMENT OF SUBSTITUTE PRODUCTS OR SERVICES, OR FOR ANY INDIRECT, SPECIAL, INCIDENTAL, EXEMPLARY OR CONSEQUENTIAL DAMAGES OF ANY KIND ARISING OUT OF OR IN CONNECTION WITH USE OF THE MASSARRAY SYSTEM AND ANY HARDWARE, SOFTWARE, CONSUMABLES, COMPONENTS, AND DOCUMENTATION PROVIDED TO CUSTOMER BY AGENA, HOWEVER CAUSED AND REGARDLESS OF THE THEORY OF LIABILITY (WHETHER IN CONTRACT, TORT (INCLUDING NEGLIGENCE), STRICT LIABILITY OR OTHERWISE). AGENA’s TOTAL AND CUMULATIVE LIABILITY ARISING UNDER OR IN CONNECTION WITH CUSTOMER’S USE OF THE MASSARRAY SYSTEM AND ANY HARDWARE, SOFTWARE, CONSUMABLES, COMPONENTS, AND DOCUMENTATION, WHETHER IN CONTRACT, TORT (INCLUDING NEGLIGENCE), STRICT LIABILITY OR OTHERWISE, SHALL BE LIMITED AND SHALL NOT EXCEED THE AMOUNT RECEIVED BY AGENA FROM CUSTOMER PURSUANT TO PURCHASE ORDER AND INVOICE OR WRITTEN CONTRACT WITH CUSTOMER. THE LIMITATIONS SET FORTH IN THIS SECTION SHALL APPLY REGARDLESS OF FORESEEABILITY AND EVEN IF AGENA OR ITS SUPPLIERS HAVE BEEN ADVISED OF THE POSSIBILITY OF SUCH LOSSES, COSTS, OR DAMAGES. THE FOREGOING SHALL APPLY REGARDLESS OF ANY FAILURE OF ESSENTIAL PURPOSE OF ANY LIMITED REMEDY.

FRM-CUS-018 Rev03


Appendix F Warranty70



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