USG CUS 071...• Updated Troubleshooting Appendix. • Removed reference to iPLEX Gold. R01 9/28/15...

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

iPLEX® Pro and Gold Reagents USER GUIDE

iPLEX® Pro and Gold Reagents User GuideDOC. USG-CUS-071 Rev034/17/19

Copyright 2015-2019. 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 USA.

TRADEMARKSMassARRAY, iPLEX, Agena Bioscience, and SpectroCHIP are registered trademarks 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; 9,310,378; 9,394,565; 9,669,376; and 9,896,724, 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. [0818]

Revision HistoryRevision Number Date Changes Made

R03 4/17/19 • Corrected typo in PCR Primer Mix initialconcentration in Table 3.1 and Table C.1.

• Removed instrument instruction chapters—referto instrument user guides.

• Removed reference to 24-pad format.

• Updated Table 1.1, Agena Chemistriesdescription.

• Updated Table 1.4 (iPLEX Reagents), Table 1.5(Instruments and Equipment), Table 1.6 (OtherRequired Reagents and Labware).

• Clarified language in Table 1.8 (Lab AreaActivities).

• Clarified language on DNA, reagent, and analytestorage conditions in Section 1.5 (ImportantProcedures and Guidelines).

• Changed water type in PCR reaction (to HPLC-grade) to be consistent with other reactions.

• Corrected SAP Buffer final concentration amountin Table 3.2 and Table C.3.

• Added description of iPLEX Gold in Chapter 1.

R02 5/24/16 • Updated resin, water addition, and DNA amountrequirements.

• Updated Troubleshooting Appendix.

• Removed reference to iPLEX Gold.

R01 9/28/15 Initial release.

USG-CUS-071 Rev03iPLEX® Pro and Gold Reagents User GuideFor Research Use Only. Not for use in diagnostic procedures.

Contents

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

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

Workflow Overview . . . . . . . . . . . . . . . . . . . . 4

Inventory Checklist . . . . . . . . . . . . . . . . . . . . 6

Laboratory Work Areas . . . . . . . . . . . . . . . . . . . 10

Important Procedures and Guidelines . . . . . . . . . . . . . . 11

Customer Support . . . . . . . . . . . . . . . . . . . . 12

Chapter 2 Setting up the Experiment in the Software. . . . . . . . . . . . 13

Designing PCR and Extension Assays . . . . . . . . . . . . . . 13

Importing the Assay Design File with Typer AssayEditor . . . . . . . . 13

Creating a Virtual Plate in Typer PlateEditor . . . . . . . . . . . . 14

Chapter 3 Assay Protocol . . . . . . . . . . . . . . . . . . . . . . . 17

Adjusting the Extension Primers . . . . . . . . . . . . . . . . 17

Preparing DNA from Samples. . . . . . . . . . . . . . . . . 18

Performing PCR Amplification. . . . . . . . . . . . . . . . . 19

Performing SAP Treatment . . . . . . . . . . . . . . . . . 20

Performing the iPLEX Extension Reaction. . . . . . . . . . . . . 21

Water Addition. . . . . . . . . . . . . . . . . . . . . . 23

Chapter 4 Analyzing Data . . . . . . . . . . . . . . . . . . . . . . . 25

Viewing Results in TyperAnalyzer . . . . . . . . . . . . . . . 25

Generating Reports in TyperAnalyzer . . . . . . . . . . . . . . 27

Report Types . . . . . . . . . . . . . . . . . . . . . . 28

Appendix A Extension Primer Adjustment Methods . . . . . . . . . . . . . 29

Overview . . . . . . . . . . . . . . . . . . . . . . . 29

Two-Tier Method . . . . . . . . . . . . . . . . . . . . . 29

Three-Tier Method . . . . . . . . . . . . . . . . . . . . 30

Four-Tier Method . . . . . . . . . . . . . . . . . . . . . 31

Regression Method . . . . . . . . . . . . . . . . . . . . 32

Appendix B Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . 35

ii


Appendix C Quick Reference Guide: Reaction Conditions . . . . . . . . . . 41

PCR Reaction Conditions. . . . . . . . . . . . . . . . . . . 41

SAP Reaction Conditions. . . . . . . . . . . . . . . . . . . 41

iPLEX Extension Reaction Conditions . . . . . . . . . . . . . 42

Appendix D Additional Peaks . . . . . . . . . . . . . . . . . . . . . . 43

Appendix E Licensing . . . . . . . . . . . . . . . . . . . . . . . . . 45

Appendix F Warranty. . . . . . . . . . . . . . . . . . . . . . . . . . 49


Chapter 1

Overview and Inventory Checklist

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Inventory Checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Laboratory Work Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Important Procedures and Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Customer Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.1 Introduction

This user guide provides instructions for performing SNP genotyping and somatic mutation analysis using iPLEX® Pro or iPLEX Gold reagents on the MassARRAY® System and MassARRAY Typer application software. The iPLEX extension reaction is a universal method for detecting insertions, deletions, substitutions, and other polymorphisms in amplified DNA.

iPLEX Gold provides a robust genotyping chemistry for agricultural applications while iPLEX Pro provides the high performance and sensitivity required for pharmacogenetics, inherited disease and somatic variant assays.

This user guide covers iPLEX Pro and Gold chemistry only. See Table 1.1 for an overview of Agena Bioscience chemistries. For more information on iPLEX HS and UltraSEEKTM chemistries, consult the iPLEX HS Panels User Guide, and the UltraSEEK Panels User Guide, both available on Agena Cx.com, or contact Agena Bioscience Customer Support.

iPLEX reagents and the MassARRAY System are for research use only and not for use in diagnostic procedures.

Chapter 1 Overview and Inventory Checklist4


Table 1.1 Agena Bioscience Chemistries

1.2 Workflow Overview

Following assay design, extension primer adjustment, and sample preparation, multiplexed PCR is performed, using at least 10 ng of input DNA per well (Figure 1.1). After PCR, any remaining free deoxynucleotides in the amplification reaction mixture must be dephosphorylated to prevent interference with the iPLEX extension reaction. Shrimp alkaline phosphatase (SAP) dephosphorylates unincorporated dNTPs and converts them to inactive dNDPs, making them unavailable for future reactions.

iPLEX reaction cocktail (primer, enzyme, buffer, terminator nucleotides) is then added to the amplification products and the mixture is thermocycled to process the iPLEX reaction, which involves the enzymatic addition of terminator nucleotides into the diagnostic site. In the reaction mixture, all four terminator nucleotides—A, T, C, and G—are present. During the iPLEX reaction, the primer is extended by one of the nucleotides, which terminates the extension of the primer. Using a DNA polymerase that incorporates nucleotides, the iPLEX

Chemistry Limit of Detection

Markers per Reaction

Applications Sample Types

iPLEX Pro 5% - 10% < 40 • Pharmacogenetics

• Inherited variants

• Crop strain validation

• Somatic variants with high minor allele frequency

• Blood

• Buccal cells

• Hair follicles

• Ear punches

• WGA DNA

• FTA cards

iPLEX Gold 5% - 10% < 40 • Livestock testing

• Biomarker validation

• Blood

• Buccal cells

• Hair follicles

• Ear punches

• WGA DNA

• FTA cards

iPLEX HS > 1% < 15 • Somatic mutations

• Low-frequency variants

• NGS discovery validation

• GMO screening

• Formalin-fixed, paraffin embedded tissue (FFPE)

• Fine need aspirates (FNA)

• Cytology blocks

• Core need biopsy

• Seed mixtures

UltraSEEK > 0.1% < 10 • Oncogenic resistance and progression monitoring

• Plasma

• Circulating cell-free DNA (ccfDNA)

• Circulating tumor DNA (ctDNA)

• Circulating tumor cells (CTCs)

Chapter 1 Overview and Inventory Checklist 5


reaction produces allele-specific extension products of different masses depending on the sequence analyzed (see Table 1.2 for example).

Table 1.2 Example of Extension Products of a Heterozygote Assay

The iPLEX reaction products are then desalted and transferred onto a SpectroCHIP Array, which is analyzed in the MassARRAY Analyzer. Data acquired by the MassARRAY Analyzer is processed by MassARRAY Typer software and reports are generated that detail the mutation/genotype calls.

Workflow steps are shown in Table 1.3.

Table 1.3 Workflow Steps

Analytes Peak descriptionLength of

product [bp]Calculated mass [Da]

Unextended Primer

Extension Primer 20 6163.0

Allele 1 Extension Primer + A 21 6434.2

Allele 2 Extension Primer + G 21 6450.2

Software Setup See...

Design assays (PCR and extension primers) with Assay Design Suite software.

Section 2.1 and

Assay Design Suite User Guide

Import the assay design file into Typer Assay Editor software. Section 2.2

Create a virtual plate in Typer Plate Editor software. Section 2.3

Assay Protocol See...

Adjust the extension primers. Section 3.1

Prepare DNA from samples. Section 3.2

Amplify DNA using the user-designed PCR primers. Section 3.3

Perform SAP treatment to dephosphorylate any remaining free dNTPs. Section 3.4

Process the extension reactions using the user-designed Extend primers.

Section 3.5

Data Acquisition and Analysis See...

Desalt the extension products, transfer to a SpectroCHIP Array, and analyze on the MassARRAY Analyzer.

Applicable instrument user guide (available on AgenaCx)

View data and generate reports. Chapter 4



1.3 Inventory Checklist

Required Agena Bioscience Reagents

Table 1.4 lists the required Agena Bioscience reagents needed to perform iPLEX reactions. iPLEX reactions may be performed in 96- or 384-well formats. Upon receipt, store the items as described.

Figure 1.1 iPLEX chemistry



Table 1.4 iPLEX Reagents

Required Instruments Table 1.5 Instruments and Equipment

Materials Provided Shipping ConditionStorage

Temperature

Storage Location (see Table 1.8 Lab

Areas)

PCR Reagent Set

• MgCl2• 10X PCR Buffer

• dNTP Mix

• PCR Enzyme

Dry Ice -10 to - 25°C Lab Area 2

iPLEX Reagent Kit (Pro or Gold)

• iPLEX Pro Enzyme or iPLEX Enzyme

• 3-Pt. Calibrant

• iPLEX Termination Mix

• iPLEX Buffer Plus (10X)

• SAP Buffer

• SAP Enzyme

Dry Ice -10 to - 25°C Lab Area 2

SpectroCHIP Arrays Ambient Temperature

Room Temperature

Lab Area 3

Clean Resin Ambient Temperature

Room Temperature

Lab Area 3 (paraffin-cover Clean Resin)

Instruments and Equipment Specifications

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

Table 1.6 Other Required Reagents and LabwareItem Supplier

PCR oligos Any. Assay Design Suite generates a file ("DesignName_order.xls") which lists the PCR oligos to order. These should be ordered suspended at 100 μM with no special purification required (standard, de-salted). Alternatively, they can be ordered lyophilised and be resuspended in sterile HPLC-grade water at 100 μM.

Extend oligos Any. Assay Design Suite generates a file ("DesignName_order.xls") which lists the Extend oligos to order. These should be ordered suspended at 500 μM with no special purification required (standard, de-salted). Alternatively, they can be ordered lyophilised and be resuspended in sterile HPLC grade water at 500 μM.

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, 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 and 50 mL; sterile

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

Pressure pads

HPLC-grade water Residue after evaporation ≤2ppm;Sterile, nuclease- and DEPC-free;>18.2 MΩ

Ethanol Absolute (200 proof)

Tween 20 (optional; only needed for Nanodispenser RS1000 troubleshooting)

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



WARNING!WARNING! The Chip Prep Module is configured at installation for the particular plates you will be using. If at any point you wish to change the plates you are using, contact Agena Bioscience Customer Support to update instrument configuration. Using plates with different well depth without reconfiguration can compromise assay performance.

See MassARRAY System: Recommended Lab Equipment and Set-up, available on AgenaCx.com (Product Support > Applications & Systems > MassARRAY System), for more detailed information on recommended lab set up and recommended vendors.

Required Software Table 1.7 Required Software

For 96-format experiments:

96-well full-skirted, low-profile microtiter plates (MTPs)

OR

96-well non-skirted microtiter plates

Max fill volume: 0.2 mL

Working volume: 0.2 mLMax fill volume: < 0.3 mL

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:

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

Item Supplier

Software Supplier

MassARRAY v4.1.83 or higher• Assay Editor. Imports assay design

files to the MassARRAY database. See page 13.

• PlateEditor. Creates a virtual plate that represents the physical plate with the analytes (sample plate). See page 14

• TyperAnalyzer. Displays the spectral results data in table and graph formats and generates results reports. See page 25.

Agena Bioscience (AgenaCx.com: Product Support > Application Updates > Typer)

Assay Design Suite Software Hosted online by Agena Bioscience. Users log into the host website via AgenaCx.com (AgenaCx.com > Online Tools).

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

http://cran.r-project.org

See MassARRAY Typer Release Notes on AgenaCx.com (Product Support > Application Updates > Typer) 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, 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

MassARRAY Analyzer software

• Chip Linker. Links the virtual plate created in Typer Plate Editor with the associated SpectroCHIP Array.

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

• 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 sample plate.

2Pre-PCR preparation, including preparation of the PCR cocktails and addition of PCR cocktails to the reaction plate. Preparation of the SAP and extension cocktails.

3

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



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.

• 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.

• Pulse vortex 5 times and centrifuge tubes and plates with reagents before use or cycling.

• Seal plates with plate 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.

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

• Store the reagents according to labeling when not in use.

• At designated stopping points in the assay protocol, finished reactions in plates can be stored after thermocycling: at 4°C for up to 24 hours, or at -20°C for more than 24 hours. Do not store for more than 2 weeks.

• 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: 1-858-882-2800Fax: 1-858-882-2727Help Desk: 1-877-4-GENOME or (+1) 858-882-9300E-mail: [email protected]

EUROPE Agena Bioscience GmbHGasstrasse 18, House 522761, Hamburg GermanyPhone: (+49) 40-899676-0Fax: (+49) 40-899676-10Email: [email protected]

ASIA PACIFIC Agena Bioscience, Inc.Ground Floor, 27 Jeays StreetBowen Hills, QLD 4006 AustraliaPhone: (+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 Road, Xuhui DistrictShanghai 200032 PR ChinaPhone: (+86) 21 6427 0566Fax: (+86) 21 6427 0511Email: [email protected]

[0419]


Chapter 2

Setting up the Experiment in the Software

Designing PCR and Extension Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Importing the Assay Design File with Typer AssayEditor . . . . . . . . . . . . . . . . . . . . . . . . . . 13Creating a Virtual Plate in Typer PlateEditor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

2.1 Designing PCR and Extension Assays

Use Agena Bioscience’s online tool, Assay Design Suite (ADS) (AgenaCx.com > Online Tools), to design genotyping and somatic mutation assays for use with the MassARRAY System. ADS provides full support for human, bovine, and mouse genome sequences, and allows users to:

• Import target sequences from a list of rs numbers, FASTA files, or sets of formatted SNP sequences.

• Check design criteria.

• Generate information and documents for oligo ordering, downstream workflow, and assay performance predictions.

See Assay Design Suite Genotyping and Somatic Mutation User Guide, available at AgenaCx.com (Product Support > Documentation > SNP Genotyping) for more information.

2.2 Importing the Assay Design File with Typer AssayEditor

The assay design file generated by the Assay Design Suite software when you created your custom assay design 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 an assay design file:

1. Open Typer AssayEditor.

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

Chapter 2 Setting up the Experiment in the Software14


3. 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.

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

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

6. Click the Browse button next to Assay Group.

7. Navigate to the folder that contains the files created by Assay Design Suite.Choose the file with the suffix “AssayDesign.xls” or “AssayDesign.txt” and click Open.

8. Click the Import button to import the file.

2.3 Creating a Virtual Plate in 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:

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

• Create a virtual sample plate.

• Apply assays to the plate.

• Apply 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

Chapter 2 Setting up the Experiment in the Software 15


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.

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.

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 (24-, 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.

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 Add Samples from Group or Add Sample.

4. Select File > Save from the toolbar.

Chapter 2 Setting up the Experiment in the Software16


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Chapter 3

Assay Protocol

Adjusting the Extension Primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Preparing DNA from Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Performing PCR Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Performing SAP Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Performing the iPLEX Extension Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Water Addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

3.1 Adjusting the Extension Primers

Because of the inverse relationship between peak intensity and analyte mass, as well as the occurrence of non-predictable variations in peak heights (due to inconsistent oligonucleotide quality or poor desorption/ionization in the mass spectrometer), extension primers in iPLEX assays must be adjusted by concentration before running your experiment. The adjustment method involves analyzing each extension primer on the MassARRAY System and generating a primer adjustment report, which identifies extension primers that need to be adjusted before you proceed with your genotyping experiment.

IMPORTANT!Adjusting the extension primers is critical to successful multiplexing and requires the use of a SpectroCHIP Array. If the extension primer concentration is not adjusted, an assay with a very low intensity primer peak will systematically fail when applied to samples as part of a multiplex.

The extension primer mix varies somewhat depending on whether a low plex or high plex reaction is being processed. Extension primer adjustment methods are described in detail in Appendix A.

1. For each extension primer:

a. Prepare a primer mix using one of the methods in Appendix A.

b. Prepare a primer mix sample by pipetting 1 L of the primer mix into a well of a non-skirted microtiter plate and adding HPLC-grade water (24 L for a 384-well plate; 49 L for a 96-well plate), to obtain a 360 nM dilution.

2. Repeat Step 1 for each extension primer mix to generate a sample plate that contains primer mix samples for each of your multiplexed assays.

3. Dispense the primer mix samples to a SpectroCHIP Array using standard dispensing conditions for iPLEX reaction products, as described in Section 4.2, Section 5.2, or Chapter 6 (depending on the particular MassARRAY System components you are using). It is recommended you dispense to three pads per primer mix.

If the entire SpectroCHIP Array is not used, you may keep it for future use in adjusting iPLEX primer mixes. However, do not reuse previously spotted pads. Store the SpectroCHIP Arrays in their original packaging in a desiccator. SpectroCHIP Arrays may be stored for one week maximum.

Chapter 3 Assay Protocol18


4. Create a plate in MassARRAY PlateEditor, and add the assay definitions, including a water or NTC sample. See Section 2.3 or the MassARRAY Typer 4 User Guide for detailed instructions.

5. Analyze the SpectroCHIP Array in the MassARRAY Analyzer, as described in Section 4.3, Section 5.3, or Chapter 6 (depending on the particular MassARRAY System components you are using).

6. Perform a quality check on the extension primers and the extension primer mixes by reviewing the spectra. Each well on the SpectroCHIP Array will yield no-calls because there is no analyte, only unextended extension primers. A peak should appear at the expected mass for each extension primer in the mix. A missing peak generally indicates poor primer quality or a primer missing from the mix. An unexpected peak generally indicates poor primer quality or the addition of an unnecessary primer to the mix.

7. Generate a Primer Adjustment Report in MassARRAY Typer Analyzer (File > Reports > PrimerAdjustment Report) to determine if any of the primer mixes need adjusting.If all of the peaks are at least 45% of the height of the highest peak, they are acceptable. If any peak is less than 45% of the height of the highest peak, add more of that primer. Column F in the report indicates the amount to add, as a fraction of the given primer’s original volume.

NOTEAdjust the original primer mix, not the primer mix sample in the microplate.

3.2 Preparing DNA from Samples

IMPORTANT!Perform this procedure in Lab Area 1.

Types of Samples iPLEX genotyping can be performed on genomic DNA or cDNA isolated from a variety of samples, including fresh tissue, frozen tissue, formalin-fixed paraffin-embedded (FFPE) tissue, cell lines, blood plasma, serum, buccal cells, and hair follicles.

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.

Amplicons selected by Assay Design Suite are typically 70-150 bp in length. Since the amplicons are relatively short, only genomic DNA is discussed in this section—in most cases, this information is also applicable to cDNA. If you wish to use cDNA templates, it is assumed you have a cDNA generation method already optimized.

Chapter 3 Assay Protocol 19


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.

Amount iPLEX genotyping and somatic mutation requires at least 10 ng of DNA per well. Prepare a working dilution of at least 5 ng/L; 2 L of this dilution will be added to each well. Note that at least 1500 amplifiable copies are needed. The genomic DNA may be assessed for quality and quantity of amplifiable copies using Agena Bioscience’s iPLEX Pro Sample ID Panel.

The minimum/maximum amount of template DNA should be confirmed empirically by running a pilot experiment.

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.

3.3 Performing PCR Amplification

You will amplify genomic DNA from your samples using your custom-designed PCR primers in a final reaction volume of 5 L.

The PCR cocktail tables in this section are designed for high-plex levels such as a 24-plex.

IMPORTANT!Perform this procedure in Lab Area 2. 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.



Table 3.1 PCR Cocktail

*For more than one reaction, an overhang should be added to the volumes. The recommended amount for manual dispensing is 20%,. Automated dispensing may require a higher overhang; contact Agena Bioscience for information on purchasing automation reagent kits with higher fill volumes.

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

3. Dispense 3 μL of PCR cocktail to each well of a new PCR reaction plate.

4. Seal the PCR reaction plate and centrifuge at 3200 x g for 1 minute.

5. Visually inspect the individual wells from the bottom of the PCR reaction plate to confirm uniform and adequate cocktail solution is present in every well before continuing.

6. Dispense 2 μL of DNA (5 ng/μl) to each well of the PCR reaction plate. Make sure that pipette tips do not come into contact with the PCR cocktail already in the plate to avoid cross-contamination.

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

8. Perform thermocycling using the following conditions:

3.4 Performing SAP Treatment

IMPORTANT!You will perform the SAP addition 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.

ReagentInitial

Concentration

Final concentration

(in 5 μl)1 well (μl)*

HPLC-grade water n/a n/a 0.8

10X PCR Buffer 10X 1X 0.5

MgCl2 25 mM 2 mM 0.4

dNTP Mix 25 mM 500 M 0.1

PCR Primer Mix 500 nM 100 nM 1.0

PCR Enzyme 5 U/l 0.2 U/l 0.2

PCR Cocktail Volume 3.0

95°C 2 minutes

95°C 30 seconds

56°C 30 seconds 45 cycles

72°C 1 minute

72°C 5 minutes

4°C Hold



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.

Table 3.2 SAP Cocktail



4. Dispense 2 μL of SAP cocktail into each well 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. Visually inspect the individual wells from the bottom of the reaction plate to confirm uniform and adequate cocktail solution is present in every well before continuing.

7. Perform thermocycling using the following 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.

3.5 Performing the iPLEX Extension Reaction

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

Preparing iPLEX Extension Reaction Cocktail IMPORTANT!

Prepare the iPLEX extension reaction cocktail in Lab Area 2. 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 SAP-treated reaction plate at 3200 x g for 5 seconds.

ReagentInitial

Concentration

Final concentration

(in 7 l)1 well (l)*


SAP Buffer 10X 0.24X 0.17

SAP Enzyme 1.7 U/l 0.073 U/l 0.30

SAP Cocktail Volume 2.00

37°C 40 minutes

85°C 5 minutes 1 cycle

4°C Hold



2. Prepare the iPLEX extension 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 3.3.

Table 3.3 iPLEX Extension Reaction Conditions



Adding the iPLEX Extension Reaction Cocktail to the Reaction Plate

IMPORTANT!Add the iPLEX extension reaction cocktails to the reaction plate in Lab Area 3.

1. Dispense 2 L of extension reaction cocktail into each well of the reaction plate. Change tips after each dispense.

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

3. 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.

4. Thermocycle the reaction plates 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.

ReagentInitial

Concentration

Final concentration

(in 9 l)1 well (l)*


iPLEX Buffer Plus 10X 0.222X 0.20

iPLEX Termination Mix 10X 0.222X 0.20

Extend Primer Mix 5 - 15 M 0.52 - 1.57 M 0.94

iPLEX Pro Enzyme or iPLEX Enzyme

32 U/L 0.142 U/L 0.04

Extension Reaction Cocktail Volume

2.00

95°C 30 seconds

95°C 5 seconds

52°C 5 seconds5 cycles 40

cycles80°C 5 seconds

72°C 3 minutes

4°C Hold



3.6 Water Addition

IMPORTANT!Perform this procedure in Lab Area 3.

1. Add HPLC-grade water to each well of the reaction plate using a 12-channel multipipettor. iPLEX panels typically require 16 L per well for 384-well plates, and 30 L or more for 96-well plates. Contact Agena Customer Support for assistance in determining the optimal water addition amount for your panel.

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

3. Process the plate on the MassARRAY System with Chip Prep Module or MassARRAY Nanodispenser RS1000 and MassARRAY Analyzer. See the appropriate instrument user guides, available on AgenaCx.com (Applications & Systems > MassARRAY System), for instructions.

If not proceeding directly to processing the plate on the MassARRAY System, the reaction plate should be sealed, and stored at 4°C (if storing for less than 24 hours), or at -20°C (if storing for more than 24 hours). Do not store for more than 2 weeks.


Chapter 4

Analyzing Data

Viewing Results in TyperAnalyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Generating Reports in TyperAnalyzer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Report Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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.

4.1 Viewing Results in TyperAnalyzer

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

2. To select the results to display, put a check mark next to the SpectroCHIP Array in the Chip List. If more than one SpectroCHIP Array is check-marked, click on the SpectroCHIP Array name to select it. The active SpectroCHIP Array will be highlighted.

3. You may now view and manage data in a number of different panes in the TyperAnalyzer window (see Figure 4.1), allowing you to:

• Display data in table and graphical formats.

• Automatically check results for errors.

• Apply cluster analysis to results.

• Highlight results in call cluster plots according to user-specified meta-data.

• Exclude user-specified unusable results from the TyperAnalyzer windowpanes.

• Manually call a genotype.

• Restore the original genotype call.

Chapter 4 Analyzing Data26


Figure 4.1 TyperAnalyzer Default Window Layout

Traffic Light paneA plate map of the active SpectroCHIP Array. Selecting a well updates the Call Cluster Plots, Details pane, and Sample Plot.

A list of the assays on the SpectroCHIP Arrays that are check-marked. Selecting an assay updates the:• Active SpectroCHIP Array• Selected well in the Traffic

Light pane• Call Cluster Plots• Chip and Assay Summaries• Sample Plot

TyperAnalyzer displays the results from the active SpectroCHIP Array that is check-marked and highlighted. Changing the active SpectroCHIP Array updates the:• Traffic Light pane• Chip Summary• Call Cluster Plots• Post Processing

Clusters• Details pane• Plate Data pane• Sample Plot

Click a tab to view a pane:Post Processing ClustersCluster analysis (using a Gaussian mixtures approach) of the selected assay results on all SpectroCHIP Arrays that are check-marked.Call Cluster PlotResults of the selected assay on the active SpectroCHIP Array plotted in a user-selected format:• High mass peak height vs. low mass

peak height (linear or log axes)• Yield vs. skew• High mass yield vs. low mass yieldSelecting a point in a graph updates the:• Spectrum in the Details pane• Selected well in the Traffic Light paneHistogramIncludes the results of all assays on the SpectroCHIP Arrays that are check-marked. Selecting an assay in the histogram (clicking a histogram bar) updates the:• Call Cluster Plots• Details pane

Click a tab to view a pane:Automated Data Error CheckingSummary of errors detected by the software:• Positive control error• Negative control error• Mendelian error• Discordance errorChip Summary Information about the active SpectroCHIP Array.Assay Summary Information about the assays on the active SpectroCHIP Array.

Directory tree of results in the MassARRAY database.

Sample Plot Results of the selected assay in two graphs:• SNR vs. Sample ID• Height vs. Sample ID

Chapter 4 Analyzing Data 27


4.2 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 in the TyperAnalyzer menu bar.TyperAnalyzer will generate the report directly into Excel readable format, and save it in the Typer-4\bin\TyperReports folder. Some panels also generate interactive reports in HTML format; in those cases the HTML index file will open automatically in your browser.

Figure 4.2 Generating Reports

Chapter 4 Analyzing Data28


4.3 Report Types

Table 4.1 lists the different types of reports generated by TyperAnalyzer. The reports that are available depend on the type of experiment. See MassARRAY Typer 4 User Guide for more detailed information.

Table 4.1 Report typesReport Name Description

Plate Summary Provides basic information about a SpectroCHIP Array (for example, ID, date, user) and brief statistics about the number of assays, samples, plexing level, number of calls, call rate, and status of calls.

Allelotype Includes allelotyping data such as average frequency of allele 1 and 2, frequency error, assay status, primer frequency, and pause frequency.

Allelotype Correction Includes the same data as the allelotype report and also corrections for heterozygous skewing.

Assay Type Count Lists the number of assays for the selected data.

Best Call Probability Shows the best score for the same sample and assay across multiple results. The Score column in the report indicates how good the call was on a scale from 0 to 1.

Call Probability Provides the same information as the Plate Result report and includes the call for each well. The Score column indicates how good the call was on a scale from 0 to 1.

Description Count Provides a count of each call status (A. Conservative through O. Unknown genotype) for the selected data.

Genotype Area Includes genotyping data for the selected experiment(s) and additional information derived from peak area data.

Plate Definition Lists the assays and samples in each well of the plate.

Plate Result Lists the calls for each well of the plate.

Primer Adjustment Indicates the primers that require volume adjustment to compensate for the inverse relationship between analyte mass and signal-to-noise ratio.

AllPlateSummary Provides the results of the Plate Summary Report for all the selected SpectroCHIP Arrays in the Chip View in one single report.

QC Report Summarizes data and post processing results for a user-selected well, assay, and SpectroCHIP Array.


Appendix A

Extension Primer Adjustment Methods

A.1 Overview

The extension primer mix varies somewhat depending on whether a low plex or high plex reaction is being processed. The minimum recommended method for adjusting extension primers for low plex (2 plex - 18 plex) reactions is a 2-tier method, dividing the primers into a low mass group and a high mass group. For example, in a 18-plex, the 9 lowest mass primers would be at a concentration of 0.73 µM and the 9 highest mass primers would be at 1.46 µM in the final 9 µl reaction.

The minimum recommended method for adjusting high plex (19 plex - 36+ plex) extension primers is a 3-tier method, dividing the primers into a low mass group, a medium mass group, and a high mass group. For example, in a 36-plex, the 12 lowest mass primers would be at a concentration of 0.52 µM, the 12 middle mass primers would be at a concentration of 1.04 µM, and the 12 highest mass primers would be at 1.57 µM in the final 9 µl reaction.

Table A.1 Primer concentrations for uniplex, low plex, and high plex reactions

Four options are described below for preparing iPLEX primer cocktails. The first three methods involve grouping the primers into 2, 3, or 4 tiers according to mass and adjusting the concentration. The fourth method (regression) involves mixing every primer at a specific concentration according to mass and equilibrating each primer in the spectrum using specific concentrations. Agena Bioscience’s field service engineers can advise and train you on the best methods for your application.

A.2 Two-Tier Method

The two-tier method involves doubling the concentration of high and low mass primers in two groups, as shown in Figure A.1.

Uniplex Low (2-18) plex High (19-36+) plex

Low Mass 1.00 M 0.73 M 0.52 M

Medium Mass n/a n/a 1.04 M

High Mass n/a 1.46 M 1.57 M

Appendix A Extension Primer Adjustment Methods30


Prepare the 7/14 µM iPLEX extend primer mix as follows:

1. Divide the extension primers into 2 groups, LOW mass, and HIGH mass.

2. Calculate the LOW mass group for 7 µM in the final iPLEX extend primer mix.

3. Calculate the HIGH mass group for 14 µM in the final iPLEX extend primer mix.

4. Calculate the remaining water volume.

This is the easiest method to use for low plex reactions (2-18 plex). However, this may result in lower call rates for higher plex reactions. For maximal call rates for high plex reactions, use one of the following options instead.

A.3 Three-Tier Method

The three-tier method involves adjusting the concentration of high and low mass primers in three groups, as shown in Figure A.2.

Figure A.1 Two-Tier Primer Adjustment Method

Low Mass High

Figure A.2 Three-Tier Primer Adjustment Method

1st Group 2nd 3rd

Appendix A Extension Primer Adjustment Methods 31


The first group of low mass primers are mixed at 5.0 µM, the medium mass group at 10 µM, and the high mass group at 15 µM. Prepare the 5/10/15µM iPLEX extend primer mix as follows:

1. Divide the extension primers into 3 groups, LOW mass, MEDIUM mass, and HIGH mass.


3. Calculate the MEDIUM mass group for 10 µM in the final iPLEX extend primer mix.



A.4 Four-Tier Method

The four-tier method involves adjusting the concentration of high and low mass primers in four groups, as shown in Figure A.3.

The first group of low mass primers are mixed at 7.0 µM, the following group at 9.3 µM, the third group at 11.6 µM, and the higher mass group at 14.0 µM. Prepare the 7/9.3/11.6/14 µM iPLEX extend primer mix as follows:

1. Divide the extension primers into 4 groups from LOW mass to HIGH mass.


3. Calculate the two MEDIUM mass groups for 9.3 and 11. 6 µM in the final iPLEX extend primer mix.



Figure A.3 Four-Tier Primer Adjustment Method

1st Group 2nd Group 3rd Group 4th Group



A.5 Regression Method

The regression method involves mixing every primer at a specific concentration according to mass. Knowing the mass, it is possible to equilibrate each primer in the spectrum using specific concentrations. For example, from the data in Figure A.4, the following relation can be derived:

Intensity = -300.77Ln(Mass) + 2730.7

This relation does not consider oligonucleotide purity and non-predictable sequence-specific desorption/ionization variance; however, it is still useful in providing a good starting point. A sample plot is shown in Figure A.4.

A Microsoft® Excel spreadsheet can be created to calculate concentration and dilution to apply to each primer within a reaction. For example, apply the following formula to calculate the diluted oligo concentration:

(LN(Mass)-7.82) * oligo stock concentration = target primer concentration in mixture

Knowing the initial concentration and volume, additional water volume (µl) can be calculated, and an equivalent volume of diluted primers can be used to create the working mix. To prepare the extend primer mix using linear adjustment method:

1. Add additional water into each well as per the linear Primer Regression spreadsheet.

2. Pool an equal volume (15 µL) of each primer from each well into a single tube to create the Extend primer working pool.

Figure A.5A below shows a 23-plex equimolar iPLEX primer mix before adjustment. The last seven oligos, and particularly the one at ~7700 Da, have intensities close to detection limits. An iPLEX reaction on a heterozygous sample with below average yields would produce allelic peaks below detection thresholds. Figure A.5B shows the same primers mixed using the regression method.

Figure A.4 Relation of Peak Intensity and Mass

Inte

nsity

Mass (Da)

Appendix A Extension Primer Adjustment Methods 33


Figure A.5 Comparison of Non-adjusted 23-plex Primer Mix vs. Adjusted Mix Using Regression Method

A. Non-adjusted Equimolar Primer

B. Adjusted with Regression Method


Appendix B

Troubleshooting

The following tables list some potential problems and solutions that may help you troubleshoot your iPLEX experiments. More information may be found in the following user guides, all available at AgenaCx.com or by contacting Agena Bioscience Customer Support (page 12).

• 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 MassARRAY Nanodispenser and MassARRAY Analyzer

Table B.1 Troubleshooting (MassARRAY Nanodispenser and MassARRAY Analyzer)Problem Possible Reason Possible Solution

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 nanopure deionized 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 nanopure deionized 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 water solution in place of the regular water addition.

Appendix B Troubleshooting36


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 cut-off (default is 10%).

Peak intensities are too low.

Poor crystallization of analyte and matrix on a 24-pad SpectroCHIP Array; Did not wait 5 minutes for analyte/matrix to dry on a 24-pad SpectroCHIP Array.

Resdispense analyte to a new SpectroCHIP Array and wait at least 5 minutes before proceeding.

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 4.2or Figure 5.10.

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.

Problem Possible Reason Possible Solution

Appendix B Troubleshooting 37


Peaks are not aligned to the expected calls.

Calibration may not be enabled in the SpectroACQUIRE software.

Ensure calibration is enabled.

MassARRAY Analyzer is not properly tuned.


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.

Error when generating reports.

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

Problem Possible Reason Possible Solution



Troubleshooting MassARRAY System with Chip Prep Module

Table B.2 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 compressed 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 6.3 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 compressed 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 6.3 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 48).

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 43).

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.

Error when generating reports.

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

Appendix B Troubleshooting 39


Table B.3 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 C

Quick Reference Guide: Reaction Conditions

C.1 PCR Reaction Conditions

Prepare the PCR master mix 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 C.1. Add each PCR primer and DNA separately.

Table C.1 PCR Reaction Conditions

*Add an overhang of 20% for more than one reaction for manual dispensing. Automated dispensing may require a higher overhang; contact Agena Bioscience for information on purchasing automation kits with higher fill volumes.

Table C.2 PCR Thermocycling Conditions

C.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 C.3.

ReagentInitial

Concentration

Final concentration

(in 5 μL)1 well* (μL)


10X PCR Buffer 10X 1X 0.5

MgCl2 25 mM 2 mM 0.4

dNTP Mix 25 mM 500 M 0.1

PCR Enzyme 5 U/L 0.2 U/L 0.2

PCR Master Mix Final Volume 2.0

PCR Primer 500 nM 100 nM 1.0

PCR Cocktail Final Volume 3.0

DNA (5 ng/L) 2.0

PCR Reaction Final Volume 5.0

95°C 2 minutes

95°C 30 seconds

56°C 30 seconds 45 cycles

72°C 1 minute

72°C 5 minutes

4°C Hold

Appendix C Quick Reference Guide: Reaction Conditions42


Table C.3 SAP Cocktail


Table C.4 SAP Thermocycling Conditions

C.3 iPLEX Extension Reaction Conditions

Prepare the iPLEX 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 C.5. Add each Extend primer separately.

Table C.5 iPLEX Extension Reaction Conditions


Table C.6 Extension Reaction Thermocyling Conditions

ReagentInitial

Concentration

Final concentration



SAP Buffer 10X 0.24X 0.17

SAP Enzyme 1.7 U/L 0.073 U/L 0.30

SAP Cocktail Final Volume 2.00

37°C 40 minutes

85°C 5 minutes 1 cycle

4°C Hold

ReagentInitial

Concentration

Final concentration



iPLEX Buffer Plus 10X 0.222X 0.20

iPLEX Termination Mix 10X 0.222X 0.20

iPLEX Pro Enzyme or iPLEX Enzyme

32 U/L 0.142 U/L 0.04

Extension Master Mix Final Volume 1.06

Extend Primers 5 - 15 M 0.52 - 1.57 M 0.94

Extension Reaction Cocktails Final Volume 2.00

95°C 30 seconds

95°C 5 seconds

52°C 5 seconds5 cycles 40

cycles80°C 5 seconds

72°C 3 minutes

4°C Hold


Appendix D

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 D.1 shows the expected peaks from each depurination or depyrimidation event, and Figure D.1 shows a small peak at -150 from the main peak, corresponding to a guanidine depurination.

Table D.1 Expected Peaks from Depurination/DepyrimidationEvent

Depurination A -134 Da

G -150 Da

Depyrimidation C -110 Da

T -125 Da

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

Appendix D Additional Peaks44




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 Licensing46


(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 47


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 Licensing48




Appendix F

Warranty

RESEARCH USE ONLY Customer is hereby put on notice that AGENA BIOSCIENCE (“AGENA”) products, including the MassARRAY System, and all other MassARRAY System-related hardware, software, and consumables (except the MassARRAY Dx System, and all other MassARRAY Dx System-related 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 other MassARRAY System-related hardware, software, and consumables, (except the MassARRAY Dx System, and all other MassARRAY Dx System-related 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

Appendix F Warranty50


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 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

Appendix F Warranty 51


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 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 Rev04

Appendix F Warranty52



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