Ion PI IC 200 Kit - Thermo Fisher Scientifictools.thermofisher.com/content/sfs/manuals/MAN0010078_Ion_PI_IC_200_UG.pdfThe Ion PI™ IC 200 Kit (Cat. no. 4488377) contains all materials

USER GUIDE

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

Ion PI™ IC 200 Kitfor use with:Ion Proton™ SystemIon Chef™ System

Catalog Number 4488377Publication Number MAN0010078

Revision C.0

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

DISCLAIMER

TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.

Important Licensing Information

This product may be covered by one or more Limited Use Label Licenses. By use of this product, you accept the terms and conditions of all applicableLimited Use Label Licenses.

Trademarks

All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Microsoft, Windows, and Excel aretrademarks of Microsoft Corporation. Macintosh is a trademark of Apple, Inc. Guava and easyCyte are trademarks of Guava Technologies, Inc. Cy is atrademark of GE Healthcare UK Limited. Agencourt and Ampure are trademarks of Beckman Coulter, Inc. Bioanalyzer is a trademark of AgilientTechnologies, Inc. ELGA and PURELAB are trademarks of VWS Limited. Rainin, Pipet-Aid, and Pipet-Lite are trademarks of Rainin Instrument, LLC.MAXYMum Recovery is a trademark of Axygen, Inc. Tygon is a trademark of Saint-Gobain Performance Plastics Corporation. Orion is a trademark ofThermo Orion, Inc. Kimwipes is a trademark of Kimberly-Clark Corporation.

©2015 Thermo Fisher Scientific Inc. All rights reserved.

Contents

About this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Revision history . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Prerequisites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

About the kit and related products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9About the Ion PI™ IC 200 Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Library compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Kit contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Ion Proton™ Wash 2 Bottle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Ion Chip Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Required materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

About the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Ion Chef™ Instrument components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Interior hardware and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17About the Ion Chef™ System touchscreen interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

About the Ion Proton™ Sequencer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Ion Proton™ Sequencer components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Ion Proton™ Sequencer reagent positions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19About the Ion Proton™ Sequencer touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Precautions - Read before using the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Avoid nucleic acid contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Avoid chip damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Guidelines for using Ion Chef™ reagents and consumables . . . . . . . . . . . . . . . . . . . . . . 21

Precautions - Read before using the Ion Proton™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Gas safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Avoid nucleic acid contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Avoid CO2 contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Avoid introducing noise to instrument measurements . . . . . . . . . . . . . . . . . . . . . . . . . . 22Avoid chip damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Ion PI™ IC 200 Kit User Guide 3

■ CHAPTER 2 Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Create a Planned Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24About Planned Runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Create a Planned Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Planned Run wizard key fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Dilute the libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Prepare the Ion Chef™ System for use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

■ CHAPTER 3 Run the Ion Chef™ System .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

Set up the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Prepare the libraries and consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Prepare the chip for loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Load the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Ion Chef™ pre-run checklist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Start the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

■ CHAPTER 4 Clean and initialize the Ion Proton™ Sequencer . . . . . . . 50

Guidelines for handling and inserting chips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Insert a chip into the chip clamp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Prepare 1 M NaOH daily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52Cleaning schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5218 MΩ water cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Chlorite cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Initialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Initialization guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58Begin the initialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58Prepare and install the Wash 1 and Wash 3 Reagent Tubes . . . . . . . . . . . . . . . . . . . . . 58Prepare the Wash 2 Bottle and install . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Prepare and install the Reagent Tubes with dNTP solutions . . . . . . . . . . . . . . . . . . . . . 60

■ CHAPTER 5 Start the sequencing run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Unload and prepare the chips for sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Sequence the chip on the Ion Proton™ Sequencer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Contents

4 Ion PI™ IC 200 Kit User Guide

■ CHAPTER 6 Clean the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

About the cleaning protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Clean the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Ion Chef™ System setup and operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Ion Proton™ Sequencer alarms and events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Ion Proton™ Sequencer status bar icon warnings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Instrument leaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Instrument error messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Sample loading or sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

ISP Quality Control assay troubleshooting table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

■ APPENDIX B Supplementary procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Quality control of Ion PI™ IC 200 Ion Sphere™ Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Quality Control assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Quality control using the Qubit® 2.0 or Qubit® 3.0 Fluorometer . . . . . . . . . . . . . . . . . . 89Quality control using the Guava® easyCyte™ 5 Flow Cytometer . . . . . . . . . . . . . . . . . . . 90Quality control using the Attune® Cytometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Quality control using the Qubit® 2.0 Fluorometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Upgrade the Qubit® 2.0 Fluorometer firmware and software . . . . . . . . . . . . . . . . . . . . 92Upgrade for firmware V2.00 or V3.00 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93Upgrade for firmware V3.10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95Calculate the Qubit® 2.0 Fluorometer Calibration Factor . . . . . . . . . . . . . . . . . . . . . . . . 97Measure the templated unenriched sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100Acceptance criteria for unenriched Ion Sphere™ Particles . . . . . . . . . . . . . . . . . . . . . 103

Quality control using the Qubit® 3.0 Fluorometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104Calculate the Qubit® 3.0 Fluorometer Calibration Factor . . . . . . . . . . . . . . . . . . . . . . . 104Measure the templated unenriched sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108Acceptance criteria for unenriched Ion Sphere™ Particles . . . . . . . . . . . . . . . . . . . . . 111

Maintain the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112Install a firmware update . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112Change the instrument name . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113

Contents


■ APPENDIX C Ion Chef™ and Torrent Server network setup . . . . . . . 114

Networking overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114About the Ion Chef™ instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115About the Torrent Server . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115Networking guidelines and best practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115Network setup workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115Network setup examples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115Collect the required network information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116General requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

Ion Chef™ Networking Configuration Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118Target the Torrent Server IP Address . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

Editing the Torrent Server firewall tables to allow the LAN port IP . . . . . . . . . . . . . . . . . . 125Create an exception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

Enable Planned Run sharing among Torrent Servers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127Set up Server Network (admin action) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Transfer a Planned Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

Undo a Planned Run transfer (administrator) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Undo a Planned Run transfer (user) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

■ APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

Symbols on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133Conformity symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Location of safety labels on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

Safety information for instruments not manufactured by Thermo Fisher Scientific . . . . 135

Instrument safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136Physical injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136Electrical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137Cleaning and decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137Ultraviolet (UV) Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Safety and electromagnetic compatibility (EMC) standards . . . . . . . . . . . . . . . . . . . . . . . . . 137Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138EMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138Environmental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

Contents


About this guide

CAUTION! ABBREVIATED SAFETY ALERTS. Hazard symbols and hazardtypes specified in procedures may be abbreviated in this document. For thecomplete safety information, see the “Safety” appendix in this document.

IMPORTANT! Before using this product, read and understand the information in the“Safety” appendix in this document.

Revision history

Revision Date Description

C.0 6 April 2015 • Updated for new Graphical User Interfaceinstrument firmware downloaded with TorrentSuite™ Software v4.4.

• Updated for use of the Qubit® 3.0 Fluorometer forquality control of templated ISPs (see “Qualitycontrol using the Qubit® 3.0 Fluorometer“ onpage 104).

• New Ion Chef™ and Torrent Server networkingappendix added (see Appendix C, “Ion Chef™ andTorrent Server network setup“).

B.0 15 October 2014 • Updated library input recommendations.

• Ion Chef™ pre-run checklist added (page 44).

• Minor corrections and edits.

A.0 19 May 2014 Initial release, which includes instructions to set up andoperate the Ion Chef™ System with the Ion PI™ IC 200 Kit(Cat. no. 4488377).


Purpose

This user guide describes how to use the Ion Chef™ System to prepare enriched,template-positive Ion Sphere™ Particles (ISPs) for up to 200 base-read sequencing oflibraries on the Ion Proton™ Sequencer. The Ion PI™ IC 200 Kit (Cat. no. 4488377)includes reagents and materials sufficient for performing 4 dual-sample, template-preparation runs on the Ion Chef™ System and subsequent sequencing on an IonProton™ System.

WARNING! The protection provided by the equipment may be impaired if theinstrument is operated outside the environment and use specifications, the userprovides inadequate maintenance, or the equipment is used in a manner notspecified by the manufacturer (Life Technologies).

Prerequisites

This guide also assumes that you have:• A general understanding of Ion Torrent™ sequencing chemistry and workflow• Knowledge of techniques for handling and preparing DNA libraries

About this guidePurpose


Product information

About the kit and related products

The Ion PI™ IC 200 Kit (Cat. no. 4488377) includes the reagents and materials requiredto use the Ion Chef™ System to prepare template-positive Ion Sphere™ Particles forsequencing. The kit also includes reagents and materials for loading on Ion PI™ v2 BCChips for sequencing the prepared template using the Ion Proton™ Sequencer.

The Ion PI™ IC 200 Kit can be used with up to 200-base-read libraries of any typeprepared using any available Ion library kit.

The Ion PI™ IC 200 Kit is compatible with Torrent Suite™ 4.4 and later. Be sure toupdate your Torrent Server to the latest available version of Torrent Suite™ beforeusing this kit.

Note: If you are currently using version 4.2.1 of the Ion Reporter™ Software with anearlier version of the Torrent Suite™, contact Technical Support for assistance withupgrading to Torrent Suite™ 4.4.

1

About the Ion PI™

IC 200 Kit

Librarycompatibility

Softwarecompatibility


Kit contents and storage

IMPORTANT! Do not substitute components from any other Ion sequencing kits. LifeTechnologies has validated this protocol using these specific materials. Substitutionmay adversely affect system performance.

IMPORTANT! Store all consumables and cartridges under the recommendedconditions and in an upright position.

The Ion PI™ IC 200 Kit (Cat. no. 4488377) contains all materials required for templatepreparation and sequencing in the boxes shown below. Upon arrival, inspect allconsumables and contact Life Technologies if any of the products have been damagedduring shipping.

Ion PI™ IC 200 Kit summary

Ion PI™ IC 200 Kit (Cat. no. 4488377)

Component Part number Quantity perkit

Ion PI™ IC Supplies 200 4488378 4 boxes

Ion PI™ IC Solutions 200 4488380 4 cartridges

Ion PI™ IC Reagents 200 4488379 4 cartridges

Ion Proton™ Sequencing Supplies 200 4488651 1 box

Ion PI™ IC Sequencing Reagents 200 4488381 1 box

Ion PI™ IC Sequencing Solutions 200 4488652 1 box

Ion Proton™ Chip Preparation Materials:

• Ion Proton™ Chip Adapter

• Ion PI™ Chip Preparation Solution

• Ion Chip Priming Solution

4485416

4488159

A25604

8 adapters

2 tubes

2 tubes

Chapter 1 Product informationKit contents and storage1


Ion Chef™ reagents and materials

Ion PI™ IC Supplies 200 (Part no. 4488378; 4 boxes supplied per kit)

Component Quantity perbox Storage

Chip Adapter 2 adapters

15° to 30°C

Enrichment Cartridge 1 cartridge

Ion PI™ IC 200 Tip Cartridge 1 cartridge

PCR plate 1 plate

PCR plate seal 1 plate seal

Recovery Station Lid 2 lids

Recovery Tube 12 tubes

IC Piercing Tips 2 tips

Ion PI™ IC Solutions 200 (Part no. 4488380)

Component Quantity Storage

Ion PI™ IC Solutions 200 Cartridge 4 cartridges 15° to 30°C

Ion PI™ IC Reagents 200 (Part no. 4488379)


Ion PI™ IC Reagents 200 Cartridge 4 cartridges -30° to -10°C

Ion Proton™ Sequencer reagents and materials

Ion Proton™ Sequencing Supplies 200 Kit (Part no. 4488651)


Ion Proton™ Reagent Tube Caps 16 caps

15° to 30°CIon Proton™ Wash Bottle Sippers 4 sippers

Ion Proton™ Reagent Tube Sippers 32 sippers

Ion Proton™ Reagent Tubes w/ labels 8 tubes

Chapter 1 Product informationKit contents and storage 1


Ion PI™ IC Sequencing Reagents 200 Kit (Part no. 4488381)

Component Cap color Quantity Volume Storage

Ion PI™ dGTP Black 1 tube 180 µL

-30° to-10°C

Ion PI™ dCTP Blue 1 tube 180 µL

Ion PI™ dATP Green 1 tube 180 µL

Ion PI™ dTTP Red 1 tube 180 µL

Ion PI™ IC Sequencing Solutions 200 Kit (Part no. 4488652)

Component Quantity Volume Storage

Ion PI™ IC W2 Solution 2 bottles 125 mL

2°C to8ºCIon PI™ IC 1X W3 Solution 2 bottles 125 mL

Ion Cleaning Tablet 4 tablets —

Ion Proton™ Chip Preparation Materials

Part no. Component Quantity Volume Storage

4485416 Ion Proton™ Chip Adapter 8 adapters — Ambient

4488159 Ion PI™ Chip Preparation Solution 2 tubes 1.3 mLeach

-30° to-10°C

A25604 Ion Chip Priming Solution:

• 10X Chip Prime Solution

• Chip Prime Dilution Buffer

1 tube

1 tube

320 µL

1.5 mL

-30° to-10°Cuntil

opened,then at2°C to

8ºC

Chapter 1 Product informationKit contents and storage1


Ion Proton™ Wash 2 Bottle

The Ion Proton™ Wash 2 Bottle is sold separately and may be used for up to 20 IonProton™ Sequencer initializations (40 runs).

Contents Quantity Catalog no. Shipping andstorage

Ion Proton™ Wash 2 Bottle with label (2 L) 1 bottle A24893 15° to 30°C

Ion Chip Kit

The following Ion Chip Kit is compatible with the Ion PI™ IC 200 Kit, and is soldseparately:

Contents Quantity Catalog no. Storage

Ion PI™ Chip Kit v2 BC 8-Pack (2 boxes[1] of4 chips each)

8 chips 4484270 15° to 30°C

[1] Part no. 4484269

Chapter 1 Product informationIon Proton™ Wash 2 Bottle 1


Required materials and equipment

The following tables list required materials and equipment that are not provided withthe Ion PI™ IC Sequencing Reagents 200.

IMPORTANT! Life Technologies has verified this protocol using these specificmaterials. Substitution may adversely affect system performance.

Required materials and equipment for sequencing

3 Description Supplier Catalog no. Quantity

Ion Proton™ System (instrument and server) and allincluded accessories

LifeTechnologies

4476610 1

Ion Chip™ Minifuge (120 V or 230 V) LifeTechnologies

4479672 (120V)or

4479673 (230V)

1

Ion Proton™ Rotor and Buckets Kit LifeTechnologies

4482578 1

Ion PI™ Controls Kit 200 [1] LifeTechnologies

4488985 1

Tank of compressed nitrogen (grade 4.5, 99.995% pure orbetter)

MajorLaboratory

Supplier (MLS)

— —

Multistage (dual-stage) gas regulator (0–50 psi, 2–3 Baroutput)

VWRInternational

55850-422 1 or 2

(Optional) 1/8" x 1/4" stem reducing coupler (onlyrequired if using a separate tank for the wash station)

McMaster 5779K699 1 per wash

Uninterruptable Power Supply (UPS) [2] MLS — 1

ELGA® PURELAB® Flex 2 Water Purification System or

ELGA® PURELAB® Flex 3 Water Purification System or

Equivalent 18 MΩ water system

LifeTechnologies

4474525 or

4474524 or

MLS

1

NaOH (10 M) molecular biology grade MLS — —

Isopropanol (100%) MLS — —

Nuclease-free water molecular biology grade MLS — —

0.22-µm or 0.45-µm vacuum filtration system and filters MLS — —

Microcentrifuge[3] MLS — 1

P2, P10, P20, P200, P1000 µL pipette set and filtered tips MLS — 1 set

25-mL or 50-mL serological pipettes or

100-mL graduated cylinder

MLS — Varies

Chapter 1 Product informationRequired materials and equipment1



(If using serological pipettes) Pipet-Aid® pipetter DrummondScientific

4-000-110 1

Rainin® Pipet-Lite® XLS with RFID LTS 2 µL to 20 µL Rainin L-20XLS 1

Rainin® Pipet-Lite® XLS with RFID LTS 10 µL to 100 µL Rainin L-100XLS 1

Rainin® StableRak™ LTS tips 2-20 µL Rainin SR-L20F —

Rainin® StableRak™ LTS tips 200 µL Rainin SR-L200F —

0.2-mL MAXYMum Recovery® Thin Wall PCR Tubes, FlatCap (do not use polystyrene tubes)

Axygen PCR-02-L-C Varies

1.5-mL or 1.7-mL microcentrifuge tubes MLS — Varies

Glass bottles (1 L) MLS — —

Ice buckets and ice — — —

Aluminum heat block, capable of maintaining 50°C MLS — —

Vortex mixer with a rubber platform MLS — 1

Thermal cycler with a heated lid MLS — 1

Standard laboratory vacuum line or vacuum pump MLS — 1

Liquid trap MLS — 1

Tygon® tubing [4] MLS — 1

[1] For installation and troubleshooting.[2] For laboratories that experience frequent power outages or line voltage fluctuations, we recommend that you use an uninterruptable power

supply that is compatible with 2500 W output or higher.[3] Must fit standard 1.5- and 0.2-mL microcentrifuge tubes and generate 15,500 × g.[4] As needed to connect laboratory vacuum to liquid trap and liquid trap to P200 pipette tip.

Optional materials and equipment used to verify and adjust the W2 Solution pH duringinitialization


Orion® 3-Star Plus pH Benchtop Meter Kit with electrode,electrode stand, and calibration buffers (or equivalent)

LifeTechnologies

1112003 1

1 N HCl MLS — Varies

Magnetic stirrer (must hold 2-L bottle) MLS — 1

Magnetic stir bar (4 cm) MLS — 1

Squirt bottle MLS — 1

Chapter 1 Product informationRequired materials and equipment 1


About the Ion Chef™ System

The Ion Chef™ System provides automated, high-throughput template preparationand chip loading for use with the Ion Proton™ Sequencer. The system includes acomplete set of cartridge-based consumables and reagents that enable a user to load aIon PI™ v2 BC Chip in approximately 14.5 hours with less than 15 minutes of hands-on time. The Ion Chef™ System features network integration with the Torrent Suite™

to enable sample and reagent traceability throughout the chip preparation workflow.

The following figure illustrates the major external and internal features of the IonChef™ Instrument.

1 3

54 7 86

2

1 Door – Provides access to the interior ofthe instrument. The door is locked in theclosed position during operation.

2 Micropipettor – A mechanical positive-displacement pipettor that performs allfluid transfers during sample and chippreparation.

3 Robotic arm – Performs fluid transferduring chip loading. The arm alsocontains an optical sensor that reads thebarcodes of instrument reagents andconsumables.

4 Touchscreen – Provides access to allinstrument functions for operation,maintenance, and troubleshooting.

5 Power button – Power switch for the IonChef™ Instrument, where the states areon (illuminated) and off.

6 Power port – A 100–240 VAC port thatprovides power to the Ion Chef™

Instrument.7 Ethernet port – An RJ45 port that provides

Ethernet (100Mbit) communication withthe Ion Chef™ Instrument.

8 USB port – Provides USB communicationwith the Ion Chef™ Instrument. Used toupdate the instrument firmware and totransfer data during service ormaintenance.

Ion Chef™

Instrumentcomponents

Chapter 1 Product informationAbout the Ion Chef™ System1


The following figure illustrates the interior of the Ion Chef™ Instrument and describesthe stations involved in the preparation of Ion chips.

2 41

87

3

6

5

9

1 New pipette tips – The position of the rackcontaining unused pipette tips.

2 Automated heated cover – Transfers theplate cover to the PCR reaction platewithin the sample block. During thermalcycling, the heated cover appliescompression to seal the reaction plateand heats the cover to preventcondensation.

3 Thermal cycler sample block – Performsthermal cycling of the sequencingreactions on a 96-well PCR reaction plate.

4 Waste pipette tips – The position of therack containing waste (used) pipette tips.

5 Reagents station – The position on theinstrument deck of the diluted libraries,NaOH, and the Ion PI™ IC Reagents 200.

6 Solutions station – The position on theinstrument deck of the Ion PI™ ICSolutions 200, which is maintained atroom temperature.

7 Chip-loading Centrifuge – Performscentrifugation of Ion chips that have beenmounted to chip-loading adapters andloaded with template-positive ISPs.

8 Enrichment station – The position of therack containing consumables for theenrichment of the template-positive ISPs.

9 Recovery Centrifuges – Twin stations thatperform centrifugation of the ISPs duringthe recovery phase of templatepreparation.

Interior hardwareand consumables

Chapter 1 Product informationAbout the Ion Chef™ System 1


The Ion Chef™ System features a simple interface for loading chips, cleaning theinstrument, and performing system maintenance and configuration tasks.

1

53 4

2

1 Set up Run button – Set up the Ion Chef™

template preparation and chip-loadingroutine. Choose Step by Step to have theinstrument lead you stepwise throughinstallation of reagents and consumables,or choose Quick Start to proceed if youhave already installed the consumables.

2 Open Door button.3 Notifications button – View notifications

about instrument status during andbetween runs.

4 Quick Start button – Proceed directly tothe Quick Start instrument setup mode.User verifies the loading of a new pipettetip cartridge and an empty pipette tip rackto hold waste tips generated during therun, before proceeding to Deck Scan.

5 Settings button – Advance to a screenproviding the following options:

• Notifications: view notifications aboutinstrument status during and betweenruns

• Instrument Settings: view currentsettings and network configuration,set instrument name, adjust time zone

• Service tools: access screens forservice-related maintenance anddiagnostics

• Torrent Server: add and manageTorrent Server connections

• Clean Ion Chef: proceed directly to theinstrument cleaning routine

• Check for updates: check availabilityof system software updates

About the IonChef™ Systemtouchscreeninterface

Chapter 1 Product informationAbout the Ion Chef™ System1


About the Ion Proton™ Sequencer

The Ion Proton™ Sequencer performs automated, high-throughput sequencing oflibraries loaded onto Ion PI™ v2 BC Chips using the Ion Chef™ Instrument.

3 4 5

9

1

2

8

6 7

1 Reagent compartment – Contains all IonProton™ Sequencer reagents necessaryfor sequencing.

2 Chip clamp – Secures the Ion PI™ v2 BCChip throughout the sequencing run.

3 Touchscreen – Provides access to allfunctions including operation,maintenance, and troubleshooting.

4 Power button – Power switch for thesequencer, where the states are on(illuminated) and off.

5 Gas port – A low-pressure port thatprovides compressed nitrogen gas to thesequencer.

6 Ethernet port – An RJ45 port that providesEthernet (100Mbit) communication withthe sequencer.

7 USB port – A USB port that provides serialcommunication with the sequencer foruse during service and maintenance.

8 Power port – A 100–240 VAC port thatprovides power to the sequencer.

9 Power switch – Power switch for thesequencer, where the states are on (O)and off (–).

1

6

3

7

4

8

5

9

10

2

1 Wash 1 Solution (W1)2 Wash 2 Solution (W2)3 Wash 3 Solution (W3)4 Clean 1 Solution (C1)5 Clean 2 Solution (C2)

6 dGTP Solution (R1)7 dCTP Solution (R2)8 dATP Solution (R3)9 dTTP Solution (R4)

10 Waste Container

Ion Proton™

Sequencercomponents

Ion Proton™

Sequencerreagent positions

Chapter 1 Product informationAbout the Ion Proton™ Sequencer 1


The Ion Proton™ Sequencer features a simple interface for loading Ion chips, cleaningthe instrument, and performing system maintenance and configuration tasks.

11 12109876

1 2 3 4 5

1 Clean button – Touch to clean theinstrument before each run.

2 Initialize button – Touch to initialize theinstrument before each run.

3 Run button – Touch to start thesequencing routine (loading, calibration,and sequencing).

4 Options button – Touch to open theInstrument Settings menu used toperform maintenance and configurationtasks.

5 Tools button – Touch to perform service-related actions:

• Auto pH – Adjusts and checks the pHof the Wash 2 Bottle solution.

• Chip Cal – Runs the chip calibrationportion of the Run program.

• Data Mgnt – Manually delete run dataor transfer data.

• Noise Screen – Provides real-timemeasurement of electrical noisereadings on the chip.

• Pressure Cal – Calibrates the gaspressure.

• Reagent Check – Measures the pH ofall reagents on the instrument.

• Screen Cal – Calibrates thetouchscreen.

• Shut Down – Shuts down or rebootsthe instrument.

6 Chip Status icon – Alerts you to the chipstatus where states include: Ready ( ),Online ( ), Imaging ( ), Sleeping ( ),and No chip detected ( ).

7 Instrument status – Touch to view a list ofalarms and events for the instrument. Thenumber of alarms and events areindicated next to the associated labels onthe button.

8 Gauges button – Toggles the presence ofthe instrument temperature data(items 9-12).

9 Instrument gas pressure ( ) – Indicatesthe instrument gas pressure (10.50 psiduring cleaning, initialization, andsequencing).

10 Chip compartment temperature ( ) –Indicates the chip compartmenttemperature (35°C when the lid is closed).

11 Disk space usage ( ) – Indicates thepercent of space used on the internaldrives.

12 On-instrument analysis ( ) – Indicatesthat on-instrument analysis is in progress( indicates no analysis).

About the IonProton™

Sequencertouchscreen

Chapter 1 Product informationAbout the Ion Proton™ Sequencer1


Precautions - Read before using the Ion Chef™ System

IMPORTANT! A primary source of contamination is spurious DNA fragments fromprevious sample processing steps. Do not introduce amplified DNA into the librarypreparation laboratory or work area.

IMPORTANT! Handle nucleotides carefully to avoid cross-contamination. Alwaysdiscard gloves after removing used Sippers from the sequencer in order to avoidcross-contamination of the nucleotides. Always discard gloves after handlingconcentrated dNTP stocks. Barrier tips are required for all dNTP pipetting steps.

IMPORTANT! To avoid possible damage to the chip due to electrostatic discharge,ground yourself before picking up a chip or placing a chip on a surface such as a labbench. For example, touch the metal frame of the bench before inserting or removinga chip from a Chip Adapter.

To ensure the proper function of the Ion Chef™ System, handle the associatedconsumables and reagents according to the following guidelines:

• Store all Ion Chef™ System consumables and cartridges under the recommendedconditions and in an upright position.

• Inspect all Ion Chef™ System consumables and cartridges for damage uponarrival and again before use.

• Hold sequencing chips by gently, gripping them by their edges.• When the Ion Chef™ System is not in use, remove all consumables and reagents

from the deck and close the instrument door.• Except for the New Pipette Ion PI™ IC 200 Tip Cartridge, do not reuse any of the

Ion Chef™ System consumables or reagents. After each run, the empty Pipette IonPI™ IC 200 Tip Cartridge is transferred to the waste tip station.

IMPORTANT! All Ion Chef™ Instrument components are single-use only.

• Use only Life Technologies kits and supplies with the Ion Chef™ Instrument. Theuse of third-party reagents and supplies can adversely affect the performance ofthe Ion Chef™ Instrument and chips prepared.

• Always load two of the same model of sequencing chips into the Ion Chef™

System. The instrument cannot load different models of sequencing chips duringthe same run.

• Sequence chips within 1 hour after the Ion Chef™ System finishes loading them. Ifyou cannot sequence a loaded chip immediately, store it within a chip storagecontainer at 4°C until you are ready to run it (up to 6 hours maximum).

Note: If you choose to store a loaded chip, remove the chip from storage at least20 minutes prior to running it, allowing the chip to warm to room temperature.

Avoid nucleic acidcontamination

Avoid chip damage

Guidelines forusing Ion Chef™

reagents andconsumables

Chapter 1 Product informationPrecautions - Read before using the Ion Chef™ System 1


Precautions - Read before using the Ion Proton™ System

WARNING! Ion instrumentation should be installed and operated in a well-ventilated environment as defined as having a minimum airflow of 6–10 airchanges per hour. Assess the need for ventilation or atmospheric monitoring toavoid asphyxiation accidents from inert gases and/or oxygen depletion, andtake measures to clearly identify potentially hazardous areas through trainingor signage. Please contact your Environmental Health and Safety Coordinatorto confirm that the Ion instruments will be installed and operated in anenvironment with sufficient ventilation.

IMPORTANT! A primary source of contamination is spurious DNA fragments fromprevious sample processing steps. Do not introduce amplified DNA into the librarypreparation laboratory or work area.


IMPORTANT! Dry ice (solid CO2) should be kept away from areas where buffers,wash solutions or sources of molecular biology grade water for the Ion Proton™

System are used. High air concentrations of subliming CO2 may influence the pH ofsuch buffers during or after their preparation. The stability of the pH of these buffersis a critical factor in the performance of the Ion Proton™ System.

IMPORTANT! Install the Ion Proton™ System on a bench that is free from vibrationsand that is not in contact with freezers, pumps, or other equipment that can causevibrations. Significant vibration during sequencing may add noise and reduce thequality of the measurements.

See the Ion Proton™ System Site Preparation Guide (Pub no. 4478733) for Ion Proton™

System space requirements and clearances.

IMPORTANT! To avoid possible damage to the chip due to electrostatic discharge,ground yourself before picking up a chip or placing a chip on a surface such as a labbench. For example, touch the metal trim on the chip compartment before inserting orremoving a chip from the chip clamp.

Gas safety

Avoid nucleic acidcontamination

Avoid CO2contamination

Avoid introducingnoise toinstrumentmeasurements

Avoid chip damage

Chapter 1 Product informationPrecautions - Read before using the Ion Proton™ System1


Before you begin

■ Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

■ Create a Planned Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

■ Dilute the libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

■ Prepare the Ion Chef™ System for use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Workflow

The following workflow illustrates how to prepare and load samples using the IonPI™ IC 200 Kit and Ion Chef™ System for sequencing on the Ion Proton™ Sequencer.

Create a Planned Run within the Torrent Suite™ Softwareand set the run parameters.

q

Dilute the library samples, prepare the Ion chips, and thaw the reagents.

q

Load the Ion Chef™ Instrument with treated Ion chips,consumables, reagents, and libraries.

q

Start the Ion Chef™ Instrument.

q

Clean and initialize the Ion Proton™ Sequencer.

q

Unload the Ion Chef™ Instrument.

q

Load the Ion Chips into the Ion Proton™ Sequencerand begin the sequencing run.

2


Create a Planned Run

IMPORTANT! This sequencing kit is compatible with Torrent Suite™ 4.4 and later.Before proceeding, check for updates to the Torrent Suite™ and Ion Chef™ Systemsoftware, and install the updates if available.

Note: If you are currently using Ion Reporter™ Software v4.2.1 with an earlier versionof the Torrent Suite™, contact Technical Support for assistance with upgrading toTorrent Suite™ 4.4.

Planned Runs contain all of the settings used in a sequencing run, including thenumber of flows, kit types, barcodes used, run type (such as DNA, RNA, oramplicons), and reference file (if any). They provide a convenient way to set up yourruns. You create Planned Runs in the Torrent Browser and assign them to barcodedLibrary Sample Tubes. The Planned Runs are retrieved by the Ion Chef™ Instrumentwhen it scans the Library Sample Tubes, and by the Ion Proton™ Sequencer when youscan the barcode of the Ion Chef™-prepared sequencing chip you are loading. Each ofthe two chips prepared in an Ion Chef™ run needs a Planned Run created for it.

Note: For more information, see Torrent Suite™ Software Documentation (v4.4),available from the Ion Community (http://ioncommunity.lifetechnologies.com/community/products/torrent_suite).

1. Open the Torrent Browser for the Torrent Server connected to your Ion Chef™

System.

2. Select the Plan tab, click Templates, locate the application that you want to run(such as AmpliSeq™ Exome, then click either:

• Plan New Run to plan a new run using the generic template for the selectedapplication.

About PlannedRuns

Create a PlannedRun

Chapter 2 Before you beginCreate a Planned Run2


http://ioncommunity.lifetechnologies.com/community/products/torrent_suite


• Plan Run in the drop-down menu under the "Gear" tab to the right of anexisting template you select from the template list.

3. In the Planned Run wizard, review the IonReporter and Application tabs andmake selections appropriate to your run. In the Kits tab, make the followingselections:

a. Select Ion Chef, then select the Ion PI™ IC 200 Kit from the Template Kitdrop-down list.

b. Select Ion PI™ IC 200 Seq Kit from the Sequencing Kit drop-down list.

c. Select the appropriate chip type in the Chip Type drop-down list.

d. Select or edit the optional information fields appropriately for your run.

Chapter 2 Before you beginCreate a Planned Run 2


e. Click Next.

Note: For a complete description of the fields of the Torrent Browserapplication, see the Torrent Browser User Interface Guide, available from the IonCommunity (http://ioncommunity.lifetechnologies.com/community/products/torrent_suite).

4. Review the Plugins and Projects tabs and make selections appropriate to yourrun.





5. In the Plan tab, enter or scan the barcodes of the Ion Chef™ Library Sample Tubesinto the appropriate Sample Tube Label fields. To specify two sample names,enter "2" in the Number of chips field and then click the check mark button to theright of this field.

Note: For a complete description of the fields of the Torrent Browserapplication, see the Torrent Browser User Interface Guide, available from the IonCommunity http://ioncommunity.lifetechnologies.com/community/products/ torrent_suite.

6. When you have completed your selections, click Plan Run at the bottom right ofthe Plan tab screen to save the run. The run is listed on the Planned Runs pageunder the name that you specified and is automatically used by the Ion Chef™

System when the associated sample is loaded and scanned.

Chapter 2 Before you beginCreate a Planned Run 2


http://ioncommunity.lifetechnologies.com/community/products/ torrent_suite

http://ioncommunity.lifetechnologies.com/community/products/ torrent_suite

Field name Description

Application Select the sequencing application you are performing.

Barcode Set(optional)

If you are using barcodes with:

• DNA libraries: Select the IonXpress barcode set, whichincludes all barcodes in the Ion Xpress™ Barcode Adapters1‑96 Kits.

• RNA libraries prepared using the Ion Total RNA-Seq Kit v2:Select the IonXpressRNA barcode set, which contains all16 barcodes in the Ion Xpress™ RNA BC01-16 Kit(Cat. no. 4475485).

If you are not using barcodes with:

• DNA libraries: Leave the Barcode field blank.

• RNA libraries prepared using the Ion Total RNA-Seq Kit v2:Select RNA_Barcode_None from the drop-down list. This willensure that the proper trimming is performed on theresulting sequence when the RNA library does not have abarcode.

BED files Select the Target Regions or HotSpot Regions BED file on the IonTorrent Server, if any.

Export If installed and enabled, and you want to upload data to the IonReporter™ Software, select the Ion Reporter™ Uploader.

Flows Enter the appropriate number of flows for the read length (forexample, 500 flows for 200-base-read sequencing).

Monitor Set thresholds for Bead Loading, Usable Sequence, and KeySignal. In the Torrent Browser Monitor4Runs in Progress tab, analert is displayed if the values for a run fall below the selectedthresholds.

Planned Run Name Enter a name for the run.

Plugins Select the appropriate plugins for your application.

Project Select or add a project within which to group your run data. Youcan include runs in multiple projects, and remove runs from aproject at any time.

Reference Library Select a reference library uploaded to the Torrent Server, if any.

Sample Name(Required)

Is Ion Reporter™ Uploader enabled?

• Yes – Enter each sample name and select the appropriatevalues for workflow, relation, relation role, and set ID.

• No – Scan the barcode of the library tube into the "SampleName" and "Sample Tube Label" fields for the specificsample.

Planned Runwizard key fields



Field name Description

Sample Tube Label(Required)

Scan or enter the barcodes located on the Ion Chef™ LibrarySample Tubes that you intend to use to load samples into the IonChef™ Instrument.

IMPORTANT! You must scan or enter the barcode of each IonChef™ Library Sample Tube used on the Ion Chef™ Instrument.

Note: The "Sample Tube Label" field can be in two differentlocations in the wizard depending on adaptors or library youchoose.

Sequencing Kit Select Ion PI IC 200 Seq Kit.

Template Kit Select Ion PI IC 200 Kit.

Dilute the libraries

IMPORTANT! Before proceeding, dilute the two Ion libraries to the optimal inputconcentration. The quality of your sequencing data relies greatly upon achieving thecorrect concentration of starting library.

Dilute the two (2) stock libraries with Nuclease-free Water according to the followingtable. Prepare a fresh dilution of each library before use with the Ion Chef™ System,and use the library dilutions within 48 hours.

Library Recommended concentration [1]

RNA-Seq 100 pM

TargetSeq™ Exome 50 pM

Ion AmpliSeq™ Exome 50 pM

Ion AmpliSeq™ Exome RDY 50–100 pM

Ion AmpliSeq™ Comprehensive Cancer Panel 70 pM

Human CEPH Control 200 library[2] Dilute 2 µL into 68 µL Nuclease-freeWater

[1] Recommendations are based on qPCR quantification. If libraries are quantified with a 2100 Bioanalyzer® instrument, a higher calculated concentration may need to be used for equivalent input.

[2] Obtained from the Ion PI™ Controls 200 Kit (Cat. no. 4488985).

Chapter 2 Before you beginDilute the libraries 2


Prepare the Ion Chef™ System for use

Before you use the Ion Chef™ Instrument:• Confirm that the Ion Chef™ Instrument has been cleaned following the previous

run. If not, clean the instrument before you load it with consumables.

Note: For more information on the cleaning procedure, see Chapter 6, “Clean theIon Chef™ System “.

• Condensate may collect in the empty compartments of the Reagents andSolutions stations, depending on ambient temperature and humidity conditions.Before loading consumables into the instrument for a run, inspect thesecompartments for condensation. If necessary, wipe them dry with a Kimwipes®

wipe or absorbent cloth.• Thaw the Ion PI™ IC Reagents 200 Cartridge at room temperature for 45 minutes

prior to use.

Chapter 2 Before you beginPrepare the Ion Chef™ System for use2


Run the Ion Chef™ System

■ Set up the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

■ Start the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Set up the Ion Chef™ System

In the following procedure, you will prepare the Ion Chef™ System for use by dilutingthe two Ion libraries and loading the instrument with all of the required reagents andconsumables.

• Aluminum heat block, capable of reaching and maintaining 50°C• Ion PI™ IC 200 Kit (Cat. no. 4488377)• Ion PI™ v2 BC Chips (2)• Ion PI™ Chip Preparation Solution• Ion Proton™ Chip Adapter• 100% Isopropanol• Molecular-biology grade Nuclease-free Water• P200 pipette and filtered tips• Standard laboratory vacuum line or vacuum pump• 1X Ion Chip Priming Solution (prepared daily)• Waste container

• Prepare a 1X dilution of Ion Chip Priming Solution daily.

Note: The shipped Ion Chip Priming Solution is concentrated and must bediluted 1:10 using Chip Priming Dilution Buffer before use. Prepare 200 µL perchip that you intend to run (plus additional volume to account for pipettingerror).

• Equilibrate the Ion PI™ Chip Preparation Solution to room temperature for atleast 20 minutes before use. Keep the tube tightly capped immediately before andafter use.

• Pre-heat an aluminum heat block to 50°C.• Check for updates to the Torrent Suite™ Software and Ion Proton™ Sequencer

software, and install the updates if available.

3

Materialsrequired

Before you begin


1. At least 45 minutes prior to use, unbox the Ion PI™ IC Reagents 200 Cartridgeand allow it to thaw at room temperature.

IMPORTANT! The IC Reagents 200 Cartridge must warm to room temperaturefor at least 45 minutes before use.

2. Pipet 70 µL of each diluted library (see “Dilute the libraries“ on page 29) to thebottom of the appropriate Ion Chef™ Library Sample Tube (flagged tubes).

Note: If running the Human CEPH Control 200 Library from the Ion PI™

Controls Kit v2 (Cat. no. 4482414), prepare the control library for use by diluting2 µL of stock library into 68 µL Nuclease-free Water.

3. Cap and store the two diluted DNA libraries on ice until you are ready to loadthem onto the Ion Chef™ Instrument.

4. Remove all cartridges and consumables from their wrappings and boxes, thenplace them on the bench next to the Ion Chef™ Instrument.Prepare the following:

• Ion Chef™ Proton Chip Adapters (2)• Enrichment Cartridge• Ion PI™ IC 200 Tip Cartridge• PCR plate• PCR Plate Seal• Recovery Station Lids (2)• Recovery Tubes (12)• Ion PI™ IC Solutions 200 Cartridge• Ion PI™ IC Reagents 200 Cartridge (from Step 1)• IC Piercing Tips (2)

IMPORTANT! Prior to use, gently tap the IC Reagents 200 and the IC Solutions200 Cartridges on the bench to force the reagents to the bottoms of the tubes.

Note: When stored under normal conditions, a precipitate can form in sometubes of the IC Reagents 200 Cartridge. If present, load the cartridge as directed –the precipitate will dissolve when the reagents are mixed during instrumentoperation.

IMPORTANT! Wear gloves for the following steps. The Ion PI™ Chip PreparationSolution is a skin irritant.

1. Label a new Ion PI™ v2 BC Chip to identify the experiment, then place the chipon a stable surface such as a benchtop.

Note: The shipped Ion Chip Priming Solution is concentrated and must bediluted 1:10 using Chip Priming Dilution Buffer before use.

Note: If the chip was not used to initialize the Ion Proton™ Sequencer, skip thenext step.

Prepare thelibraries andconsumables

Prepare the chipfor loading

Chapter 3 Run the Ion Chef™ SystemSet up the Ion Chef™ System3


2. If the chip was used to initialize the Ion Proton™ Sequencer, inject 100 µLisopropanol into the chip loading port, then inject 100 µL of Nuclease-free Waterinto the loading port and remove the expelled liquid from the opposite port.Repeat the water flush one more time and remove the expelled liquid.

3. Attach an Ion Proton™ Chip Adapter to the chip exit well (located on the side ofthe chip with the notched corner, opposite the loading port; see figure below).Press firmly to ensure the adapter is tightly engaged around the well.

Chip notch

4. Perform the chip wash:a. Inject 200 µL of 100% isopropanol into the chip loading port, then remove

the expelled liquid from the exit well fitted with the adapter.

b. Attach a P200 pipette tip to a vacuum line, insert the pipette tip into the chiploading port, and aspirate the isopropanol from the chip flow cell for5-10 seconds. Inspect the chip to make sure it is dry.

Note: Make sure that the Ion PI™ Chip Preparation Solution has completelyequilibrated to room temperature before proceeding to the next step.Immediately re-cap the solution tube after use to prevent evaporation andexposure to air.

c. Inject 100 µL of Ion PI™ Chip Preparation Solution into the chip loadingport, then remove the expelled liquid from the exit well.

Note: Be careful not to introduce bubbles in the chip. If bubbles appear,repeat this wash step.

Chapter 3 Run the Ion Chef™ SystemSet up the Ion Chef™ System 3


d. Place the chip on the flat surface of an aluminum heat block pre-heated to50°C and incubate for 2 minutes.

Note: Place the chip on the side of the heat block that does not containwells, as shown in the figure below.

e. Remove the chip from the heat block and place it on a stable surface such asa benchtop.

f. Inject 200 µL of 100% isopropanol into the chip loading port, then removethe expelled liquid from the exit well. Repeat the isopropanol flush onemore time.

g. Inject 200 µL of Nuclease-free Water into the chip loading port, then removethe expelled liquid from the exit well.

h. Inject 200 µL of 0.1 M NaOH into the chip loading port, then remove theexpelled liquid from the exit well.

i. Wait 1 minute, then inject 200 µL of Nuclease-free Water into the chiploading port and remove the expelled liquid from the exit well.

5. Repeat step 4 twice more, for a total of three chip washes.

6. Inject 200 µL of 1X Ion Chip Priming Solution into the chip loading port, thenremove the expelled liquid from the exit well.

7. Attach a P200 pipette tip to a vacuum line, insert the pipette tip into the chiploading port, and aspirate the remaining fluid from the chip flow cell for5-10 seconds. Inspect the chip to make sure it is dry.

8. Remove the Ion Proton™ Chip Adapter from the chip.

(Optional) Store the prepared chip

The prepared chip can be stored for up to 24 hours before use. To store the chip:

Place the dry chip in its original anti-static blister pack and store it at roomtemperature in the dark (for example, in the box that the chip came in).



IMPORTANT! Rated centrifuge speeds are only intended for operation with providedbuckets and approved consumable chips, tubes, and sample preparation reagents.

IMPORTANT! The Chip-loading Centrifuge is rated to operate at the listed rotationalfrequencies with the chip buckets, chips, and adapters. The centrifuge must be loadbalanced. Proper care must be taken to load the bucket properly. If excessivevibrations arise, check to ensure that items are installed properly and rotors areequally balanced on each side.

IMPORTANT! Confirm that the instrument is powered ON and has been cleanedrecently following the last use.

IMPORTANT! Confirm that all components are clean and dry before loading themonto the Ion Chef™ Instrument.

IMPORTANT! Confirm that the Reagents and Solutions station compartments are dryand free of condensate before loading components.

Follow the procedure below to load the Ion Chef™ Instrument, where each stepcorresponds to a numbered area in the following diagram.

23

89

4

6

5

7

1. Open the instrument door:a. In the instrument touchscreen, touch (Open Door) then wait for the latch

to open.

Load the Ion Chef™

System



b. Lift the instrument door to the top of the travel until the latch mechanismengages.

LiftHold here

2. Load an empty pipette tip rack to the Used (Waste) Pipette Tip Position, thenchange gloves.

2

IMPORTANT! Confirm that the pipette tip rack in the Used (Waste) Pipette TipPosition does not contain any tips. The instrument will abort the run if tips arepresent in the used position.

IMPORTANT! To prevent contamination, change gloves immediately aftermoving the empty pipette tip rack to the Used (Waste) Pipette Tip Position.

3. Load a Ion PI™ IC 200 Tip Cartridge to the New Pipette Tip Position:a. Unwrap the Ion PI™ IC 200 Tip Cartridge and remove the cover to expose

the pipette tips. Load two new Ion Chef™ IC Piercing Tips into tip positionsG7 and H7 on the Ion PI™ IC 200 Tip Cartridge (numbering the positionsfrom left to right and lettering from top to bottom).

Tip positions G7 and H7 Piercing tip

1

A

2 3 4 5 6 7 8 9 10 11 12

BCDEFGH



b. Pull the catch forward, then pivot the locking bracket upwards. Load theassembled Ion PI™ IC 200 Tip Cartridge into the New Pipette Tip Position,then pull the bracket downwards to lock the cartridge in place.

3

New tip cartridge

Bracket

Catch

4. Load a new PCR plate into the thermal cycler sample block, then slide a newPCR Plate Seal underneath the automated heated cover.

IMPORTANT! When the PCR Plate Seal is positioned correctly, its tabs projectupward and contact the heated cover.

Well A1

Cover

Keyed corner4

5. Load the Ion PI™ IC Reagents 200 Cartridge and diluted libraries into theReagents station:

IMPORTANT! Thaw the IC Reagents 200 Cartridge at room temperature for45 minutes prior to use.

a. Gently tap the Ion PI™ IC Reagents 200 Cartridge on the bench to force thereagents to the bottoms of the tubes.



b. Load the cartridge into the Reagents station so that it snaps into place and islevel on the deck.

IMPORTANT! Do not force the Ion Chef™ cartridges into place. Eachcartridge fits only one location on the deck and in one orientation. If acartridge does not fit, confirm that you are loading the correct cartridge inthe correct orientation.

5

IC Reagents Cartridge

c. Uncap and load the two Library Sample Tubes, each containing 70 µL ofdiluted library, into Positions A and B on the IC Reagents 200 Cartridge.

IMPORTANT! Make sure to orient the sample tubes so that the barcodes arevisible and oriented to the right (see below).

IMPORTANT! Make sure to remove the caps to the Library Sample Tubesbefore proceeding.

d. Uncap both the tube of NaOH in Position C and the empty tube in PositionD on the IC Reagents 200 Cartridge.

D

A

B

C

00012647

00012216 Position A (DNA Library)

Position B (DNA Library)

Position C (NaOH)

Position D (Empty tube)

IMPORTANT! When the IC Reagents 200 Cartridge is loaded:

· Press down on the Library Sample Tubes to ensure that they are firmlyseated within the cartridge.

· Confirm that all tubes are uncapped, including the tube at Position D.

6. Load the Ion PI™ IC Solutions 200 Cartridge to the Solutions station:a. Gently tap the Ion PI™ IC Solutions 200 Cartridge on the bench to force the

reagents to the bottoms of the tubes.



b. Load the IC Solutions 200 Cartridge into the Solutions station until it snapsinto place and is level on the deck.

6IC Solutions Cartridge

7. Load the consumables into the Recovery centrifuges:a. Load six Recovery Tubes into each Recovery Centrifuge.

IMPORTANT! Confirm that the Recovery Tubes are seated correctly withinthe centrifuge buckets. The buckets are keyed to ensure that the tubes fit in aspecific orientation.

7

Keyed position Tube

b. Place a Recovery Station Lid over each centrifuge. Orient the lids so that theports are located as shown in the figure below.Before sealing each centrifuge, confirm that:

• The centrifuge is balanced with all required consumables.

IMPORTANT! The centrifuge must be load balanced.

• The buckets are securely seated in the centrifuge rotors.• The buckets are oriented correctly within the centrifuge so that they

pivot outwards.



c. Close the lid of the Recovery Centrifuges. Confirm that no ports are locatedin the positions facing the front of the instrument.

7a 7b 7c

No port

IMPORTANT! Do not obstruct or place any object on top of the lid.

IMPORTANT! Use only the supplied materials, including buckets anddisposables, to run the centrifuges at the rated speeds. Do not remove orchange the rotors. Inspect the buckets to assure normal operation beforeeach use.

8. Load the Enrichment Cartridge, then press down on the cartridge to ensure thatit is level with the instrument deck.

IMPORTANT! Confirm that the Enrichment Cartridge is loaded so that thelettering on the cartridge is right-side-up.

EnrichmentCartridge

Lettering

8



9. Load the Chip-loading Centrifuge:a. Load each prepared chip into a centrifuge bucket, then attach a Chip

Adapter to the assembly.

IMPORTANT! When attaching a Chip Adapter:

· Confirm that the chip has been treated with Ion PI™ Chip Prep Solutionand Ion Chip Priming Solution as described earlier.

· On the underside of each Chip Adapter, confirm that the rubber washersare in place. If a washer has become detached but can be found, returnthe washer to the empty port before you use the adapter. Otherwise,discard the Chip Adapter and use a replacement.

· Align the wells of the Chip Adapter to the wells of the chip, then gentlypush the adapter onto the chip until the clips lock into place.

· Listen for an audible 'snap', which indicates that the Chip Adapter isattached. Loading can fail if the adapter is not attached securely.

· Confirm that the tabs at all four corners of the Chip Adapter are fittedinto the slots within the centrifuge bucket.

Ports (align with chip)

Tabs

Slots

Keyed corner (align with bucket)

Chip adapter

Bucket

Ion chip

Note: If desired, you can label the tops of chips to distinguish them.

b. Load the adapter/chip/bucket assemblies into the Chip-loading Centrifuge.

Mounting grooves

Chip-loading centrifuge

9



IMPORTANT! When the Chip-loading Centrifuge is loaded, confirm thatthe Chip Adapters are firmly attached to the buckets, and that the bucketsare securely seated in the centrifuge rotors.

Chip barcode

Chip barcode

Position A marker holeChip position A

Chip position B

Note: Chip position A is 90° clockwise from the Position A marker hole.The chip loaded in this position will be loaded with ISPs prepared from thelibrary loaded in Position A of the IC Reagents 200 Cartridge. The chiploaded in Position B will be loaded with ISPs prepared from the libraryloaded in Position B of the IC Reagents 200 Cartridge.

c. Close the lid of the Chip-loading Centrifuge.

IMPORTANT! Do not obstruct or place any object on top of the lid.

Before closing the centrifuge, confirm that:• The centrifuge is balanced.

IMPORTANT! The centrifuge must be load balanced.

• The pins on the sides of the chip buckets are securely seated within thecentrifuge.

• The chip buckets are oriented correctly within the centrifuge so thatthey pivot 90° outwards when touched.

IMPORTANT! The chip buckets must be correctly seated within thecentrifuge.

IMPORTANT! The Chip-loading Centrifuge is rated to operate at the listedrotational frequencies with the chip buckets, chips, and adapters. The centrifugemust be load balanced. Proper care must be taken to load the bucket properly. Ifexcessive vibrations arise, check to ensure that items are installed properly androtors are equally balanced on each side.

IMPORTANT! Use only the materials supplied in the Ion PI™ IC 200 Kit,including buckets and disposables, to run the centrifuges at the rated speeds. Donot remove or change the rotors. Inspect the buckets prior to each use to assurenormal operation.



10. Confirm that all cartridges and reagents are installed correctly before continuing:• Confirm that each cartridge is at the correct location and in the correct

orientation.• Press down on all cartridges to confirm that they are firmly locked in place.• Confirm that all tubes on the Ion PI™ IC Reagents 200 Cartridge are

uncapped and firmly locked in place.• Confirm that the centrifuge lids are installed correctly so that the ports are

oriented toward the rear of the instrument.• Confirm that the tube and chip buckets are seated securely within the rotor

arms of the Chip-loading and Recovery Centrifuges, and that theconsumables they contain are correctly installed.

CAUTION! To ensure correct and safe instrument operation, you mustconfirm that all consumables are installed correctly to the deck before youstart a run. The Ion Chef™ Instrument does not verify all aspects of theconsumable setup prior to beginning each run.



1) A new Ion Chef™ IC 200 Tip Cartridge is loaded in the New Pipette Tip position.2) Two new Ion Chef™ IC Piercing Tips are loaded in positions G7 and H7 of the Tip Cartridge.3) Bracket is pulled downward to lock the Tip Cartridge in place.

1) A PCR Plate Seal is in place and correctly oriented under the heated cover.2) A new PCR Plate is loaded in the thermal cycler sample block.

1) The tip rack from previous run is transferred from the New Pipette Tip position to the Used Pipette Tip position.

1) IC Reagents 200 Cartridge contents are thawed (leave at RT for 45 minutes).2) IC Reagents 200 Cartridge tubes are uncapped and pressed into place at the Reagents station so that the tubes are firmly seated and level with the deck.3) Library Sample tubes are firmly seated and in the correct orientation with barcodes facing the PCR plate and caps removed.

1) IC Solutions 200 Cartridge is pressed into place at the Solutions station so that the cartridge is firmly seated and level with the deck.

1) Recovery Tubes are securely seated in the correct orientation in the Recovery Centrifuge buckets.2) Recovery Centrifuges are load-balanced.3) Recovery station lids are positioned so that the ports are correctly oriented: ports should not be located in the positions facing the front of the instrument.

1) Enrichment Cartridge is pressed into place at the Enrichment station so that the cartridge is firmly seated and level with the deck.2) Lettering on the cartridge is right-side up and positioned to the right of the enrichment tubes.

1) Chip Adapters are firmly attached to the Ion Chips.2) Adapter/chip assemblies are correctly seated in the Chip-loading Centrifuge buckets, with the adaptor clips inserted into the bucket slots.3) Keyed corner of the chip is facing inward.4) Centrifuge buckets containing the adapter/chip assemblies are securely seated in the centrifuge rotor, and freely pivot 90º outwards.5) Chip-loading Centrifuge is load-balanced.

Ion Chef™ pre-run checklist



Start the run

1. Confirm that you have loaded the instrument with all kits and consumables.

2. On the Ion Chef™ Instrument home touchscreen, touch Set up run.

3. Touch Step by Step to have the instrument lead you through the instrumentsetup, or touch Quick Start to skip the instrument setup screens.

4. Follow the on-screen instructions. When prompted, close the instrument door byfirst lifting it slightly to disengage the locking mechanism, then push down onthe door until the locks engage. After the door closes, the instrument visionsystem activates.

IMPORTANT! Do not close the door by pulling it straight down from the openposition. You must lift the door slightly before you can close it. Confirm that bothsides of the door are locked after closing it.

Lift Lower Press down

Chapter 3 Run the Ion Chef™ SystemStart the run 3


5. When prompted, touch Start check to begin Deck Scan. Wait while theinstrument scans the barcodes of all consumables and reagents to confirm theirpresence and compatibility.During Deck Scan, the touchscreen may display warnings if the Ion Chef™

Instrument detects missing or incompatible consumables. You must address allwarnings before the run can begin. After you address each condition, touch Yesto continue.

IMPORTANT! The Deck Scan function is not a substitute for manual inspectionof the reagents and consumables on the Ion Chef™ Instrument prior to starting arun. To ensure proper and safe instrument operation, confirm that allconsumables are installed correctly before you continue.

6. When Deck Scan is complete, touch Next to display the Data Destination screen.

7. Confirm that the instrument displays the correct kit name, chip types, chipbarcodes, and Planned Run. If the correct Planned Runs do not display, touch thedrop-down menu to select the Planned Run for each chip, then touch Next.

IMPORTANT! If the kit name and chip type are incorrect, confirm that you areusing the correct kit and chip. If you are using the correct kit and chip, contactTechnical Support.

Chapter 3 Run the Ion Chef™ SystemStart the run3


8. On the Run Options screen, touch the appropriate option to complete the runand enter the desired time of run completion, if necessary.

The Ion Chef™ Instrument provides two options for obtaining quality control(QC) samples that can be used to assess templating efficiency. Depending onyour selection, the QC samples will be made available either during or after therun. In either case, you can obtain unenriched samples from the correspondingLibrary Sample Tubes at Positions A and B on the Ion PI™ IC Reagents 200Cartridge, or enriched samples from Positions A and E on the EnrichmentCartridge.

By selecting… You can obtain the QC samples…

Time immediately after the run ends, at the time you specify(14.5 hours after run start).

Pause when the instrument pauses operation prior to the chiploading step (12.2 hours into the run).

Note: The DNA library in the Library Sample Tube loaded in Position A will betemplated onto ISPs that may be sampled in Position E of Enrichment Cartridgeafter a run. The DNA library in the Library Sample Tube loaded in Position Bwill be templated onto ISPs that may be sampled in Position A of EnrichmentCartridge.

9. On the Run Options screen, touch Start run to begin the run.

Note: If you need to stop the run for any reason, touch Cancel, then touch Yes toconfirm the cancellation.If the Ion Chef™ Instrument encounters a problem during the run, it will abortthe run and display the error on the instrument touchscreen. If a run fails:

a. Remove the consumables from the deck and clean the instrument. Ifpossible, retain the consumables for troubleshooting.

b. Reset and reattempt the run. If the run fails again, contact Technical Supportto troubleshoot the problem.

10. Clean and initialize the Ion Proton™ Sequencer approximately 1.5 hours beforethe Ion Chef™ System finishes chip loading.By preparing the sequencer during the last stages of chip loading, you ensurethat the chips can be sequenced as soon as possible after loading is complete.



11. If you chose to pause the run to analyze the templating efficiency, remove thesamples for testing when prompted to do so by the Ion Chef™ Instrument(approximately 12.2 hours after the start of the run).

a. When prompted to remove the QC sample, open the instrument door.

b. Transfer the unenriched QC samples (entire volume) from Positions A and Bof the Ion PI™ IC Reagents 200 Cartridge on the instrument deck to two newlabeled microcentrifuge tubes.

IMPORTANT! Do not remove the Library Sample Tubes from the Ion PI™ ICReagents 200 Cartridge.

IMPORTANT! If you unintentionally close the instrument door before youobtain the QC samples, you must wait until the end of the run before youcan collect them. You cannot pause the run or open the door once it hasbeen closed.

D

A

B

C

00012647

00012216 Position A (QC Sample)

Position B (QC Sample)

c. Analyze the QC samples.

d. Close the instrument door, then touch Continue to complete the run.

Chapter 3 Run the Ion Chef™ SystemStart the run3


12. When the run is complete, unload the Ion Chef™ Instrument and sequence thechips immediately (see “Unload and prepare the chips for sequencing“ onpage 62).

Note: If you cannot sequence a loaded chip immediately, place the chip into aseparate chip storage container and store at 4°C until you are ready to sequenceit (up to 6 hours maximum).

Note: If you are performing quality assessment of enriched samples, transfer QCsamples from positions A and E of the Enrichment Cartridge to two new labeledmicrocentrifuge tubes. See “Quality control using the Guava® easyCyte™ 5 FlowCytometer“ or “Quality control using the Attune® Cytometer“ on page 90.



Clean and initialize the Ion Proton™

Sequencer

■ Guidelines for handling and inserting chips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

■ Prepare 1 M NaOH daily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

■ Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

■ Initialization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Guidelines for handling and inserting chips

IMPORTANT! To avoid possible damage to the chip due to electrostatic discharge,ground yourself before picking up a chip or placing a chip on a surface such as a labbench. For example, touch the metal trim on the chip compartment before inserting orremoving a chip from the chip clamp.

Note: When handling chips, as a best practice, use a bare hand to touch thegrounding surface and then use the opposite hand to insert and remove chips fromthe chip clamp.

The following procedure is used at various points during cleaning, initialization, andsequencing to insert chips into the chip clamp:

1. Pull the metal tab forward to release the chip clamp.

4

Insert a chip intothe chip clamp


2. If necessary, remove the chip currently in the clamp.

3. Place the appropriate chip in the chip clamp with the chip notch in thebottom-front corner.

Note: Do not force the chip into the clamp. If the chip does not fit easily in theclamp, confirm that the notch is oriented as shown in the photo.

4. Push the metal tab back until it clicks to engage the clamp.

Prepare 1 M NaOH daily

Prepare a stock of 1 M NaOH daily by diluting 10 M NaOH with 18 MΩ water directlyfrom the purification system. Do not use water that has been collected or stored in anyother containers. You will need 32 µL for each initialization, 1 mL for each chloritecleaning, and 60 µL for each chip preparation (to prepare 600 µL of 0.1 M NaOH).

Chapter 4 Clean and initialize the Ion Proton™ SequencerPrepare 1 M NaOH daily 4


Cleaning

• 18 MΩ water (prepared and used directly from a water purification system, forexample, ELGA® PURELAB® Flex 2 Water Purification System)

• Two 140-mL Reagent Tubes (provided with kit; label the Reagent Tubes C1 andC2 before use)

• Collection tray (provided with the Ion Proton™ Sequencer)• Cleaning chip (leave chip on the instrument during cleaning)

Note: A cleaning chip is a used chip that you designate for cleaning. You can usethis chip for cleaning for up to 1 week.

• Used Sippers (from previous run or provided with the instrument)• New short blue Sippers• For chlorite cleaning only:

– Ion Cleaning Tablet (provided with kit)– 2 Reagent Tubes designated for chlorite cleaning (Relabel used C1 or C2

Reagent Tubes for this purpose)– 1 M NaOH– 0.22-µm or 0.45-µm vacuum filtration system and filters

Run the cleaning program with 18 MΩ water or chlorite solution before eachinitialization according to the following schedule. Cleaning takes ~30 minutes.

Clean with: Schedule

18 MΩ water • Before each initialization.

• (Recommended) After the last run of the day if the instrument willnot be used within 72 hours after the last run (for example, cleanafter the last run before a 3-day weekend).

• Before shutting the instrument down for an extended period.

Chloritesolution

• Once a week (unless the instrument has not been used since thelast chlorite cleaning, in which case, clean with 18 MΩ water beforeusing).

• If reagents have been left on the instrument for more than 48 hours(for example, over the weekend).

Materialsrequired

Cleaning schedule

Chapter 4 Clean and initialize the Ion Proton™ SequencerCleaning4


IMPORTANT! For the following steps, use 18 MΩ water directly from the purificationsystem. Do not use water that has been collected or stored in any other containers.

1. Select Clean on the Ion Proton™ Sequencer touchscreen Main Menu, then followthe instructions on the touchscreen to perform the cleaning procedure.

2. When prompted, orient the cleaning chip with the notch in the bottom-frontcorner, place the chip in the chip clamp, then push the metal tab back until itclicks to engage the clamp.

Note: Do not force the chip into the clamp. If the chip does not fit easily in theclamp, confirm that the notch is oriented as shown in the following photos. Formore detailed instructions, see “Insert a chip into the chip clamp“ on page 50.

3. When prompted, remove the Reagent Tubes and Wash 2 Bottle:a. Remove and discard all eight 140-mL Reagent Tubes.

Note: If necessary, you can reuse the same C1 and C2 Reagent Tubes formultiple cleanings for up to 1 week. If you reuse the C1 and C2 ReagentTubes, make sure the tubes are correctly labeled, and cap the tubes whenthey are not installed on the instrument.

b. Remove the Wash 2 Bottle, discard the liquid, and save the bottle for reusein initialization.

18 MΩ watercleaning

Chapter 4 Clean and initialize the Ion Proton™ SequencerCleaning 4


c. Remove the old Sippers from the C1 and C2 positions. Put on fresh glovesand install new short blue Sippers in those positions.

IMPORTANT! Leave all other Sippers in place; they are used duringcleaning and initialization.

4. Remove the Waste Container, empty the waste, replace the container on theinstrument, then press Next.

5. Rinse the C1 and C2 Reagent Tubes twice with ~100 mL of 18 MΩ water.

6. Add 100 mL of 18 MΩ water to the C1 and C2 Reagent Tubes and install them inthe C1 and C2 positions.



7. Place the collection tray on the instrument, direct all Sippers into the collectiontray, then press Next.

8. When cleaning is finished, press Next to return to the Main Menu.

Proceed to “Initialization“ on page 57.

Perform chlorite cleaning weekly, and as directed in “Cleaning schedule“ on page 52.

IMPORTANT! For the following steps, use 18 MΩ water directly from the purificationsystem. Do not use water that has been collected or stored in any other containers.

1. Fill a glass bottle with 1 L of 18 MΩ water and add a Cleaning Tablet (chloritetablet). Allow the tablet to completely dissolve (~10 minutes).

2. When the tablet has dissolved, add 1 mL of 1 M NaOH and filter the solutionusing a 0.22-µm or 0.45-µm filter. Use the chlorite solution within 2–3 hours anddiscard any unused solution after this time.

3. Select Clean on the Ion Proton™ Sequencer touchscreen Main Menu, then followthe instructions on the touchscreen to perform the cleaning procedure.

4. When prompted, secure a cleaning chip in the chip clamp.

5. When prompted, remove all of the Reagent Tubes and the Wash 2 Bottle:a. Remove and save the C1 and C2 Reagent Tubes for use with chlorite

solution. (Label these tubes for chlorite cleaning only and discard after thechlorite cleaning cycle is completed; do not use these tubes for 18 MΩ watercleaning.)

b. Remove and discard all other 140-mL Reagent Tubes.

c. Remove the Wash 2 Bottle, discard the liquid, and save the bottle for reusein initialization.

d. Remove the old Sippers from the C1 and C2 positions. Put on fresh glovesand install new short blue Sippers in those positions.

IMPORTANT! Leave all other Sippers in place; they are used duringcleaning and initialization.

Chlorite cleaning

Chapter 4 Clean and initialize the Ion Proton™ SequencerCleaning 4


6. Remove the Waste Container, empty the waste, replace the container on theinstrument, then press Next.

7. Add 100 mL of filtered chlorite solution to each of the two Reagent Tubesdesignated for chlorite cleaning.

8. On the Ion Proton™ Sequencer, install the tubes containing chlorite solution inthe C1 and C2 positions.

9. Place the collection tray on the instrument, then direct all Sippers into thecollection tray. Press Next to begin cleaning.

10. When cleaning is finished press Next to return to the Main Menu.

11. Remove and discard the Reagent Tubes and Sippers used for chlorite solutionfrom the C1 and C2 positions.

Note: If necessary, you can reuse the same Reagent Tubes for multiple chloritecleanings for up to 1 month. If you reuse the chlorite cleaning tubes, make surethe tubes are correctly labeled, and cap the tubes when they are not installed onthe instrument. Do not reuse chlorite cleaning tubes for 18 MΩ water cleaning.

12. Put on fresh gloves and install new short blue Sippers in the C1 and C2 positions.

13. Rinse new C1 and C2 Reagent Tubes twice with ~100 mL of 18 MΩ water.

14. Fill the C1 and C2 Reagent Tubes with 100 mL of 18 MΩ water, then install thetubes into the corresponding positions.

15. Select Clean on the touchscreen Main Menu, then press Next to advance throughthe instrument prompts until the cleaning procedure begins.

16. When the post-chlorite water rinse is complete, press Next to return to the MainMenu.

Proceed to “Initialization“.



Initialization

Initialization takes ~90 minutes.

Materials provided in the kit• 140-mL Reagent Tubes for W1, W3, and dNTP reagents

Note: Use the labels provided with the kit to label the Reagent Tubes.• Long gray and short blue Sippers for Wash Bottles and Reagent Tubes• Ion PI™ IC 200 Seq Kit dGTP, dCTP, dATP, and dTTP• Ion PI™ IC Sequencing W2 Solution• Ion PI™ IC 200 Seq Kit 1X W3 Solution

Other materials and equipment• Used chip

IMPORTANT! Initialize the instrument with a chip that has previously been usedfor sequencing. Do not use a cleaning chip.

• Ion Proton™ Wash 2 Bottle (2-L)• 18 MΩ water• 1 M NaOH (prepared fresh daily)• Ice• Filtered pipette tips and pipettes• Vortex mixer• Microcentrifuge• (If necessary to adjust pH manually) pH meter, multipoint pH calibration

reagents, pH probe, and probe stand, magnetic stirrer, stir bar, and squirt bottle


• A sequencing run should be started within 1 hour after initialization.• If you are performing more than one sequencing run per initialization, we

recommend starting the second run on the same day, but the second run may bestarted up to 24 hours after initialization.

• Replace the Reagent Tubes and Sippers every time you initialize.• Replace the Ion Proton™ Wash 2 Bottle after 40 uses (20 initializations) or

3 months, whichever comes first.• Check for updates to the Torrent Suite™ software, and install the updates if

available.

Materialsrequired

Initializationguidelines

Chapter 4 Clean and initialize the Ion Proton™ SequencerInitialization 4


Remove the dNTP stock solutions from the freezer and begin thawing on ice.

1. Remove the Sippers from the W1, W2, and W3 positions. Do not remove the usedSippers from the dNTP ports until instructed to do so.

2. Select Initialize on the touchscreen Main Menu.

3. When prompted, scan or enter the barcode on the W2 Solution bottle, or selectthe Ion PI™ IC Sequencing Reagents 200 from the drop-down list.

Note: If you are using a barcode scanner, press Enter barcode in the touchscreenbefore scanning the W2 Solution barcode.

4. Secure a used chip from an old sequencing run in the chip clamp (do not use acleaning chip), then press Next.The system verifies the gas pressure. If the gas pressure is low, press Yes to retrygas-pressure verification. If the gas pressure remains low, see “Error Message:Confirm Instrument Has Gas Pressure“ on page 76.

5. Press Next to begin the initialization.

Prepare the Wash 1 and Wash 3 Reagent Tubes.

1. Add 32 µL of 1M NaOH solution to the Wash 1 Reagent Tube.

2. Add 40–50 mL of the 1X W3 Solution from the kit to the Wash 3 Reagent Tube,measured using a serological pipette or graduated cylinder.

3. With fresh gloves, install new short Sippers in the W1 and W3 positions. Do notlet the new Sippers touch other Sippers on the instrument or any othersurfaces.

4. Install the W1 and W3 Reagent Tubes into the W1 and W3 positions of the IonProton™ Sequencer, place the collection tray beneath the dNTP Sippers, thenpress Next.

Before you begin

Begin theinitialization

Prepare andinstall the Wash 1and Wash 3Reagent Tubes

Chapter 4 Clean and initialize the Ion Proton™ SequencerInitialization4


1. Rinse the Wash 2 Bottle three times with 200 mL of 18 MΩ water, directly fromthe water purification system. Do not use water stored in other containers.

2. Extend the spigot from the water purification system into the neck of the Wash 2Bottle, then add 18 MΩ water up to the groove on the bottle (marked in thefigure below).

3. Add the entire bottle of Ion PI™ IC Sequencing W2 Solution to the Wash 2 Bottle.Immediately cap the bottle securely and invert five times to mix.

IMPORTANT! To prevent air exchange, keep the bottle tightly capped until it isattached to the Ion Proton™ Sequencer.

4. Following the on-screen prompts, use fresh gloves to install a new long Sipper inthe cap for the Wash 2 Bottle. Do not let the Sipper touch any surfaces.

Prepare the Wash2 Bottle andinstall



5. Immediately attach the prepared Wash 2 Bottle and tighten the cap. Ensure thatthe cap is screwed on tightly, then place the Wash 2 Bottle in the reagentcompartment before you continue.

6. Direct sippers to the collection tray, then press Next to continue initialization.The instrument will test the tubes for leaks, fill the Wash 1 Reagent Tube, adjustthe pH of the Wash 2 Solution, and then dilute the Wash 1 Reagent Tube solutionto the optimal concentration for the sequencing run. This procedure takes~40 minutes.

IMPORTANT! In the following steps, handle the nucleotides carefully to avoid cross-contamination and ensure that the correct dNTP solution is installed in each positionon the Ion Proton™ Sequencer.

1. After each deoxyribonucleotide (dNTP) stock solution has thawed, vortex to mixand briefly centrifuge to collect the contents. Keep dNTP stock solutions on icethroughout this procedure.

2. Use the labels provided with the kit to label four new 140-mL Reagent Tubes asdGTP, dCTP, dATP, and dTTP.

3. After the wash solutions have initialized, follow the on-screen prompts toremove the used dNTP Sippers and the collection tray.

4. Using new gloves, attach a new short Sipper to each dNTP port. Do not let theSippers touch any surfaces.

Prepare andinstall theReagent Tubeswith dNTPsolutions

Chapter 4 Clean and initialize the Ion Proton™ SequencerInitialization4


5. Using a new filtered pipette tip, carefully transfer 35 µL of dGTP stock solutioninto the bottom of the appropriate Reagent Tube, then attach the dGTP ReagentTube to the Ion Proton™ Sequencer in the correct position (front left row) andfirmly tighten.

6. Using a new pipette tip and fresh gloves for each tube, prepare and install thedCTP, dATP, and dTTP Reagent Tubes by transferring 35 µL of each dNTP stocksolution to the corresponding Reagent Tube. Make sure to install the ReagentTubes in the correct order (dGTP, dCTP, dATP, and dTTP from left to right whenfacing the instrument).

IMPORTANT! Prepare and install the reagent tubes one at a time with newgloves and pipette tips each time to avoid cross-contamination.

7. Confirm that all Reagent Tubes and Wash 2 Bottle are tightly secured, then pressNext.

• The Ion Proton™ Sequencer checks the pressure of the Reagent Tubes andWash 2 Bottle, then adds W2 Solution to each dNTP Reagent Tube.

• If a tube or bottle leaks, you will be prompted to check that it is tightlyattached to the instrument. If it continues to leak, replace it. If you replacethe tube or bottle but the instrument does not pass the leak check, contactTechnical Support.

8. At the end of initialization, the Ion Proton™ Sequencer measures the pH of thereagents.

• If every reagent is in the target pH range, a Passed screen is displayed. PressNext to return to the Main Menu. Proceed to Chapter 5, “Start thesequencing run“.

• If a Failed screen appears, see “Error message: Reagent pH: Failed; ReagentpH is displayed“ on page 82.



Start the sequencing run

■ Unload and prepare the chips for sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

■ Sequence the chip on the Ion Proton™ Sequencer . . . . . . . . . . . . . . . . . . . . . . . . . 64

Unload and prepare the chips for sequencing


to open.


LiftHold here

2. Open the lid of the Chip-loading Centrifuge, then unload both chip/bucketassemblies from the Ion Chef™ Instrument.

Remove

To unload each chip:a. Remove the chip/bucket assembly from the Chip-loading Centrifuge.

b. Apply pressure to both ends of the Chip Adapter, then remove and discardit.

5


c. Grasp the chip by its edges, carefully lift the chip out of the bucket and set itaside on a clean, static-free surface, then return the bucket to the Chip-loading Centrifuge.

Press and remove adapter

Remove carefully

Chip adapter

Bucket

Ion chip

3. Close the instrument door by first lifting it slightly to disengage the lockingmechanism, then push down on the door until the locks engage.

IMPORTANT! Do not close the door by pulling it straight down from the openposition. You must lift the door slightly before you can close it. Confirm that bothsides of the door are locked after closing it.

Lift Lower Press down

4. Load one or both chips into Ion Proton™ Sequencers and promptly begin thesequencing runs.If you cannot sequence a loaded chip immediately, place the chip into a separatechip storage container and store at +4°C until you are ready to sequence it (up to6 hours maximum).

IMPORTANT! If you choose to store a loaded chip, remove the chip from thecontainer and place it on a clean surface in the dark to warm to roomtemperature at least 20 minutes before you intend to run it.

Chapter 5 Start the sequencing runUnload and prepare the chips for sequencing 5


Sequence the chip on the Ion Proton™ Sequencer

IMPORTANT! Observe the following when sequencing the chips:

· The Ion Proton™ Sequencer must be cleaned and initialized before sequencing thechips.

· Empty the waste container before you begin the sequencing run.· Do not use reagents from other sequencing kits for sequencing chips prepared by

the Ion Chef™ System.· To avoid damage due to electrostatic discharge (ESD), do not place the chip directly

on the bench or any other surface. Always place the chip either on the Ion Proton™

Sequencer grounding plate or in the custom Ion centrifuge adapter/rotor bucket.· To avoid ESD damage, do not wear gloves when transferring chips to and from the

instrument.

Sequence the loaded chips on the Ion Proton™ Sequencer as soon as possible afterunloading the Ion Chef™ Instrument. If not, place the chip(s) into a chip storagecontainer and store at 4°C until you are ready to sequence (up to 6 hours maximum).

IMPORTANT! Do not start the sequencing run with the loaded chip. Use a used chipfor the line cleaning at the start of the run.

1. With the used chip from initialization still in the chip clamp, press Run on theMain Menu, then touch Next and confirm that "Cleaning fluid lines" displays onthe instrument touchscreen. Observe the chip for leaks. The Run Setup screenautomatically populates the fields when the Ion Proton™ Sequencer connects tothe server.

2. Follow the on-screen instructions to empty the waste bottle, load the cleaningchip, and clean the fluid lines of the Ion Proton™ Sequencer. After line cleaning,touch Next.

3. When prompted, enter or scan the barcode of the chip that you intend tosequence, then touch Next.

4. Confirm that the pre-populated settings are correct or make changes using thebuttons and drop-down lists if necessary.

Note: Note: If an error message appears, see “Error message: Not enough diskspace for the necessary number of flows“ on page 77.

5. Remove the used chip from the chip clamp and secure the chip loaded withtemplate-positive ISPs (see “Insert a chip into the chip clamp“ on page 50). Closethe chip compartment lid, then press Next to begin the sequencing run.The system calibrates the chip (~1 minute), then begins the sequencing run. Ifchip calibration fails, see “Error message: Failed: Reseat chip, then press Next torecalibrate“ on page 81.

IMPORTANT! During a run, do not open the chip compartment lid or reagentcompartment door, and avoid touching the instrument. Touching the instrumentduring the sequencing run may reduce the quality of the measurements.

Chapter 5 Start the sequencing runSequence the chip on the Ion Proton™ Sequencer5


6. If you will be sequencing a loaded chip that you have stored, remove the storedchip from the chip container in the refrigerator twenty (20) minutes before theend of the first run, and place it on a clean surface in the dark to warm to roomtemperature.

7. When the first run is complete, empty the waste container, then sequence theremaining chip as soon as possible.

IMPORTANT! After the first sequencing run is complete:

· Empty the waste container before you begin the next sequencing run.· Leave the first chip in the instrument while you initiate the second run. Load

the second chip when prompted.

When the run is complete, the touchscreen returns to the Main Menu. Use the TorrentSuite Browser to review the results.

Chapter 5 Start the sequencing runSequence the chip on the Ion Proton™ Sequencer 5


Clean the Ion Chef™ System

■ About the cleaning protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

■ Materials required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

■ Clean the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

About the cleaning protocol

The Ion Chef™ System includes an automated cleaning function that must beperformed following every run. The cleaning routine is initiated from the Ion Chef™

Instrument touchscreen and is designed to minimize potential contamination. Duringthe routine, the instrument irradiates the deck with ultraviolet light for 1 minute afterall consumables have been removed from the instrument.

IMPORTANT! Although the Ion Chef™ Instrument cleaning routine provides someprotection against contamination, it is not a substitute for good laboratory techniqueor precautions. When preparing DNA libraries for use or when preparing the IonChef™ Instrument, make certain to observe sterile laboratory procedures at all times toensure minimal contamination.

Materials required

• Gloves, powder-free nitrile• Isopropyl alcohol, 70% solution• Wipes, lint-free

Clean the instrument

IMPORTANT! Clean the Ion Chef™ Instrument as described below after every run. Toprevent contamination, do not operate the instrument unless it has been recentlycleaned.


to open.

6



LiftHold here

2. Remove and dispose of any used consumables from the instrument.

bc

gf

a

e

d

h

a. Remove and discard the PCR plate from the PCR sample block.

b. Remove and discard the box of used pipette tips from the waste tip position.

IMPORTANT! Handle the disposable reservoir in the waste tip positionwith care. During the run, liquid waste may have collected within thecontainer.

IMPORTANT! Do not reuse the waste pipette tip rack. Always move theempty Tip Cartridge from the new tip position to the waste tip position.

c. Move the empty Tip Cartridge to the waste tip position.

IMPORTANT! Do not discard the empty Tip Cartridge.

d. Remove and discard the Ion PI™ IC Reagents 200 Cartridge.

IMPORTANT! Make sure to transfer the QC samples before you removeand discard the IC Reagents 200 Cartridge.

Chapter 6 Clean the Ion Chef™ SystemClean the instrument 6


e. Remove and discard the Ion PI™ IC Solutions 200 Cartridge.

f. Close the lid of the Chip-loading Centrifuge.

g. Remove and discard the Enrichment Cartridge.

h. Remove and discard the consumables from the Recovery Centrifuges,including the:

• Recovery Station Lids• Recovery Tubes

3. Inspect the Recovery Centrifuges and clean the components if excess liquid ispresent.

Is liquidpresent? Then…

No Go to step 4.

Yes Clean the centrifuge bowl and buckets as described below.

IMPORTANT! Clean the Recovery Centrifuge occasionally, onlywhen excess liquid is noticeable in the bowl and/or buckets. Youdo not need to clean the centrifuge after every run.

To clean the Recovery Centrifuge bowl and buckets:

IMPORTANT! Wear powder-free, nitrile gloves when cleaning the RecoveryCentrifuge.

a. Remove the buckets from the Recovery Centrifuge. Clean the inside andoutside of each bucket using a lint-free wipe, then place the buckets on aclean, dry surface while you clean the centrifuge.

Bucket

Lint-free wipe

Chapter 6 Clean the Ion Chef™ SystemClean the instrument6


b. Use lint-free wipes to clean the inside rim of the centrifuge, then remove allfluid from the bottom of centrifuge bowl.

Inside rim of the centrifuge

Bottom of the centrifuge bowl

Note: When cleaning the centrifuge bowl, fold the wipe in half to provideextra absorbency.

c. Use lint-free wipes treated with 70% isopropanol to clean the:• Inside rim of the centrifuge.• Bottom of the centrifuge bowl.• Outside and inside of the centrifuge buckets.

d. Use lint-free wipes to dry the centrifuge and buckets.

e. Install the centrifuge buckets, then close the centrifuge lid.

Buckets (cleaned and installed)

4. Close the instrument door by first lifting it slightly to disengage the lockingmechanism, then pushing down on the door until the locks engage.



5. From the Ion Chef™ Instrument touchscreen, begin the cleaning:a. On the screen that appears after run completion, touch Next.

Note: You may also clean the instrument at any time starting from thehome touchscreen. Touch Settings, then touch Clean Ion Chef.

b. Confirm that you have removed all consumables from the Ion Chef™

Instrument, except for the empty Tip Cartridge in the waste tip position,then touch Next.

Chapter 6 Clean the Ion Chef™ SystemClean the instrument6


c. With the door closed, touch Start. The instrument performs a load checkbefore starting the cleaning routine. The Ion Chef™ Instrument stopsventilation and illuminates the ultraviolet (UV) light within the instrument.

CAUTION! The Ion Chef™ Instrument emits UV light at 254 nm. Wearappropriate eye wear, protective clothing, and gloves when workingnear the instrument. Do not look directly at the UV light while it isilluminated during the cleaning routine.



Troubleshooting

■ Ion Chef™ System setup and operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

■ Ion Proton™ Sequencer alarms and events . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

■ Ion Proton™ Sequencer status bar icon warnings . . . . . . . . . . . . . . . . . . . . . . . . . 76

■ Instrument leaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

■ Touchscreen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

■ Instrument error messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

■ Sample loading or sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

■ ISP Quality Control assay troubleshooting table . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Ion Chef™ System setup and operation

Observation Possible cause Recommended action

Instrument does not display thehome screen when powered on

Multiple causes 1. Power off the instrument, wait30 seconds, then power on theinstrument.

2. If the instrument fails again, contactTechnical Support.

Cannot open the instrumentdoor

An obstruction is present on oraround the door mechanism.

Remove the obstruction blocking the door,then operate the instrument normally.

Hardware or software error Contact Technical Support to report theproblem and for further assistance.

Instrument stops during a run The instrument hasencountered an internal error.

1. Record the error displayed on theinstrument display, then touch OK.

2. Contact Technical Support to report theproblem and for further assistance.

Liquid residue is present in theRecovery Centrifuge following arun

During normal instrumentoperation, a noticeable coatingof liquid can collect on the bowland buckets of the RecoveryCentrifuge following repeatedruns.

Remove the residue as instructed in “Clean theinstrument“ on page 66.

A



Instrument will not begin a run The instrument hasencountered a Deck Scan error(one or more consumables areabsent or loaded improperly).

1. Confirm that the touchscreen does notdisplay any Deck Scan warnings. If alarmsare present, note the error(s) displayed,replace the missing consumable asdirected, press No when prompted thenpress Next to cancel the run. Afterreturning to the home screen, restart therun.

2. If the error persists, confirm that:

• All buckets are seated correctly inthe rotors of the Recovery and Chip-Loading Centrifuges.

• All cartridges are loaded correctlyand are level on the instrument deck.

• The barcodes of the Ion Chef™

Library Sample Tubes are visible andpositioned correctly.

• All tubes are both present anduncapped on the Ion PI™ IC Reagents200 cartridge (Library Sample Tubes,NaOH tube, and the empty tube).

3. If the error persists after you check theconsumables on the instrument deck, doone of the following:

• If you are confident that the IonChef™ Instrument is set up correctlyand you are comfortabledisregarding the warnings, touchYES following Deck Scan to proceedwith the run.

• If the instrument cannot begin therun, contact Technical Support forfurther assistance.

The instrument hasencountered an internal error.

1. Record the error displayed on theinstrument display, then touch OK.

2. Contact Technical Support to report theproblem and for further assistance.

Appendix A TroubleshootingIon Chef™ System setup and operation A



Instrument displays one ormore alerts during a run

The instrument has detectedone or more problems duringthe run.

After the instrument completes the run,contact Technical Support.

IMPORTANT! The detected problem mightimpact the performance of the sequencing run.

• Network connection to theserver has beeninterrupted.

• User name or password isincorrect.

1. Touch the Instrument status button toview the alert(s).

2. In the Instrument status screen, confirmthat the name of the Torrent Serverconnection is red.

3. Contact your network administrator toconfirm that:

• The Torrent Server can be accessedfrom the network port used by theIon Chef™ Instrument. If not,troubleshoot the networkconnection.

• The user name and password usedby the Ion Chef™ Instrument arevalid. If not, contact the serveradministrator to renew thecredentials.

4. If the alert persists, contact TechnicalSupport for further assistance.

Chip-loading centrifugestopped unexpectedly

Centrifuge obstructed 1. Touch Open Door, then lift the door ofthe instrument.

2. Lift the lid of the centrifuge then removethe snap cover.

3. Remove the centrifuge buckets with theIon Chips.

4. If the centrifuge is obstructed, remove theobstruction.

5. Install the centrifuge buckets and IonChips in the centrifuge.

6. Snap the centrifuge cover into place, thenlower the lid.

7. Close the instrument door.8. Resume the planned run.9. If the centrifuge still does not rotate or it

makes excessive or an unusual noisewhile turning, contact Thermo FisherScientific Support.

IMPORTANT! Do not again attempt toperform the planned run on theinstrument. You may damage theinstrument.

Appendix A TroubleshootingIon Chef™ System setup and operationA


Ion Proton™ Sequencer alarms and events


Red "Alarms" and/or "Events" message in MainMenu

• Softwareupdatesavailable

• Connectivityissues

• Instrument notdetectingrequired filesor hardware

Click on the red pop-up to see detailedmessages.

• If a message states "NewerSoftware Available":

IMPORTANT! After updates areinstalled, the instrument must berestarted.

a. In the Main Menu, selectOptions4Updates.

b. Select the Released Updatescheckbox, then press Update.

c. When installation is complete,follow the onscreen promptsto restart the instrument.

Note: In some cases, theinstrument restartsautomatically after softwareinstallation.

• If a message states "NoConnectivity to Torrent Server","No Connectivity to ftp server", or"Network Manager not connected",unplug and replug the ethernetcable, confirm that the router isoperational, and verify that thenetwork is up and running.

• For any other messages:a. Power off the instrument: In

the Main Menu, selectTools4Shut Down4ShutDown.

b. Wait 30 seconds, then pressthe button on the front ofinstrument to power on theinstrument.

• If the red "Alarms" and/or "Events"message still appears in the mainmenu, contact Technical Support.

Appendix A TroubleshootingIon Proton™ Sequencer alarms and events A


Ion Proton™ Sequencer status bar icon warnings


Chip is secured in chip clamp, but chip iconindicates no chip detected

• Clamp is notengaged

• Chip is notproperlyseated

• Chip isdamaged ordirty

• Issue with chipsocket

1. Remove the chip from the chipclamp.

IMPORTANT! Do not disengagethe chip clamp if fluid is running tothe chip. If you are currentlyrunning "Clean", "Initialize", or"Run", wait until the Next buttonon the touchscreen is active, orpress Abort to return to the MainMenu before disengaging theclamp.

2. Examine the chip for damage, suchas hairline cracks, debris, or adetached flow cell.

• If the chip is damaged, inserta new chip in the chip clampand engage the clamp, look atthe chip icon to confirm thechip is detected, then pressNext or make a selection inthe Main Menu.

• If no damage can be observed,reinsert the chip in the chipclamp and engage the clamp,look at the chip icon toconfirm the chip is detected,then press Next or make aselection in the Main Menu.

Note: Alternatively, clean theback surface of the chip with alint-free laboratory wipetreated with isopropanol, thendry using a clean wipe.

3. If the chip is not detected by theinstrument, there may be aproblem with the chip socket.Contact Technical Support.

Error Message: Confirm Instrument Has GasPressure

and/or

Pressure icon indicates low gas pressure ( )

Note: The correct operating pressure is 10.5 psi.

Nitrogen gascylinder may beturned off or empty

1. Replace the gas tank if empty.2. If tank is not empty, confirm that

the cylinder has at least 500 psiand 30 psi at the outlet of theregulator. Confirm that all valvesbetween the cylinder and the IonProton™ Sequencer are open, thenpress Yes to retry verification ofgas pressure.

3. If the pressure test continues tofail, contact Technical Support.

Appendix A TroubleshootingIon Proton™ Sequencer status bar icon warningsA



Temperature icon indicates chip compartmenttemperature is out of range

Thermistor in chipcompartment isdamaged

Contact Technical Support.

Note: Do not perform sequencing runsuntil this problem is corrected; non-optimal temperatures in the chipcompartment may affect sequencing.

Error message: Not enough disk space for thenecessary number of flows

(The Ion Proton™ System hard drive does notcontain enough space for the Planned Run)

and/or

Hard drive icon indicates hard drive is almost full( )

Data normallytransferautomatically fromthe hard drive tothe Torrent Server,however this maynot happen in thecase of:

• Data transfermanuallyaborted byuser

• Issue withconnectivity ornetwork

• Incorrectconfigurationof the TorrentServer

1. Check for connectivity or networkissues, for example, unplug andreplug the ethernet cable, confirmthat the router is operational, andverify that the network is up andrunning.

2. If in "Select Planned Run", selectData Management in the touchscreen, otherwise selectTools4Data Management from theMain Menu.

3. In the Data Management screen,select All, then review the runs. Ifthere are runs that do not need tobe transferred to the TorrentServer (for example test or abortedruns), select the checkbox next tothe run names, then press DeleteSel.

4. If there are runs that you do wantto transfer, you may need to waituntil connectivity is restored forthe run to transfer and thenautodelete.

On instrument analysis icon indicates error Corrupt data filesor file system, forexample, SSD filearray is corrupted

1. Power off the instrument: In theMain Menu, select Tools4ShutDown4Shut Down.

2. Wait 30 seconds, then press thebutton on the front of theinstrument to power on theinstrument.

Appendix A TroubleshootingIon Proton™ Sequencer status bar icon warnings A


Instrument leaks


Liquid in drip pan below chipclamp

• Chip leak

• Cracked chip

• Chip clamp not closedproperly

• Leaky fluidic seal

• Problem with the chipclamp or socket

1. Press Abort to return to the Main Menu.2. Use a lab wipe to absorb the liquid in the

drip pan.3. Open the chip clamp, remove the chip,

and gently dab the chip with a lab wipe todry.

4. Look for damage to the chip, such ashairline cracks, debris, or a detached flowcell.

• If the chip is damaged, insert a newchip in the chip clamp and engagethe clamp.

• If no damage can be observed,reinsert the chip in the chip clampand engage the clamp.

5. From the Main Menu, make theappropriate selection (Clean, Initialize, orRun) to start from the beginning of theprocess.

6. If the leak persists, contact TechnicalSupport.

Leak from bottom ofinstrument

• Waste container notemptied

• Reagent tubes or bottlenot securely installed

1. Press Abort to return to the Main Menu.2. Clean up all liquid beneath the

instrument.3. Confirm that the waste container is not

overflowing, and empty if needed.4. Confirm that all Reagent Tubes and W2

Bottle are securely fastened on theinstrument.

5. If the leak is due to the waste container,Reagent Tubes, or W2 Bottle, re-start theprocedure after correcting the problem.

6. If there are no observable issues with thewaste container, Reagent Tubes, or W2Bottle, contact Technical Support.

Leak on instrument tubing(Fluid on fluid lines enteringclamp)

Fluid lines running to chipclamp are damaged or are notsecured correctly


Appendix A TroubleshootingInstrument leaksA


Touchscreen


Touchscreen not registeringinput in correct location

Touchscreen needs to becalibrated

• In Main Menu, select Tools4Screen Cal,then follow the onscreen prompts.

• If the touchscreen continues tomalfunction after Screen Cal, contactTechnical Support.

Touchscreen inoperable Damaged or defectivetouchscreen


Instrument error messages

Note: Error messages are listed in alphabetical order.


Error message: Added toomuch W1 to W2

• Poor water quality (18 MΩwater not used directlyfrom water purifier, orexposed to air for too long)

• Too much W2 Solutionused to prepare Wash 2Bottle

• Incorrect solution added tothe Wash 2 Bottle

• Too little NaOH added toWash 1 Reagent Tube

• Damaged chip

1. Confirm high water quality and correctpreparation of the 1 M NaOH and Wash 2Bottle.

2. If solution preparation is incorrect orwater quality is poor, correctly preparethe solution(s) and/or use high-qualitywater.

3. Clean the instrument.4. Repeat instrument initialization with fresh

reagents and a new (unused) chip. (Thenew chip can be used for sequencingafter initialization completes.)

Note: Once the system has added too muchNaOH, the only recourse is to clean the IonProton™ Sequencer and restart initialization orto manually adjust the pH the W2 Solution.

Error Message: Check Wash1for leaks

• W1 Reagent Tube seal isnot tight

• Tube may be damaged ordefective

1. Remove the Wash 1 Reagent Tube, thenreplace in the W1 position.

2. Make sure that the tube is securelytightened (finger-tighten).

3. If the tube continues to leak, replace thetube.

4. If leak check continues to fail, contactTechnical Support.

Appendix A TroubleshootingTouchscreen A




• Wash 2 Bottle seal is nottight

• Bottle may be damaged ordefective

1. Remove the Wash 2 Bottle, then checkthe o-ring inside the Wash 2 Bottle lid. Ifthere is any visible damage, contactTechnical Support.

2. If there is no visible damage to the o-ring,replace the Wash 2 Bottle on theinstrument. Make sure that the bottle issecurely tightened (finger-tighten).

3. If the bottle continues to leak, replace thebottle.



• W3 Reagent Tube seal isnot tight


1. Remove the Wash 3 Reagent Tube, thenreplace in the W3 position.

2. Make sure that the tube is securelytightened (finger-tighten).

3. If the tube continues to leak, replace thetube.


Error message: Chip readinginconsistent. Please replacechip and try again.

• pH response of the chip isnot uniform or reliable

• Ran out of W3 Solution orvolume too low

1. Verify that there is enough W3 Solution(approximately 50 mL) in the Wash 3Reagent Tube and that the sipper issecure.

2. If necessary, loosen the Wash 3 ReagentTube, tighten the sipper, and add moreW3 Solution to fill to 50 mL. Since thesystem pressurization gas flows when thereagent tube is loose, perform theseoperations as quickly as possible. (Thegas is not harmful to the W3 Solution andis not a hazard.)

3. If there is enough W3 Solution, replacethe chip with a new (unused) one. Securethe chip in the chip clamp, then pressStart.

Note: The new chip can be used forsequencing after initialization completes.

Error Message: ConfirmInstrument Has Gas Pressure

and/or

Pressure icon indicates low gaspressure ( )

Note: The correct operatingpressure is 10.5 psi.

Nitrogen gas cylinder may beturned off or empty

1. Replace the gas tank if empty.2. If tank is not empty, confirm that the

cylinder has at least 500 psi and 30 psi atthe outlet of the regulator. Confirm thatall valves between the cylinder and theIon Proton™ Sequencer are open, thenpress Yes to retry verification of gaspressure.

3. If the pressure test continues to fail,contact Technical Support.

Appendix A TroubleshootingInstrument error messagesA



Error message: Failed: Reseatchip, then press Next torecalibrate

(New chip fails calibration.)

• Reagents not loaded

• Clamp is not engaged

• Chip is not properly seated

• Chip is damaged or dirty

• Issue with chip socket

1. Confirm the required reagents are loadedon the instrument.

2. Press Start to re-run calibration.3. If calibration fails, remove the chip from

the clamp and look for damage to thechip, such as hairline cracks, debris, or adetached flow cell.

• If the chip is damaged, insert a newchip in the chip clamp and engagethe clamp, then press Start to re-runcalibration.

• If no damage can be observed,reinsert the chip in the chip clampand engage the clamp, then pressStart to re-run calibration.

4. If calibration fails, run reagent check:Press Abort, select Tools4ReagentCheck, then press Start.

• If the reagent check fails, re-initialize the instrument beforebeginning a sequencing run.

• If reagent check passes, contactTechnical Support.

Error message: Not enoughdisk space for the necessarynumber of flows

(The Ion Proton™ System harddrive does not contain enoughspace for the Planned Run)

and/or

Hard drive icon indicates harddrive is almost full ( )

Data normally transferautomatically from the harddrive to the Torrent Server,however this may not happen inthe case of:

• Data transfer manuallyaborted by user

• Issue with connectivity ornetwork

• Incorrect configuration ofthe Torrent Server

1. Check for connectivity or network issues,for example, unplug and replug theethernet cable, confirm that the router isoperational, and verify that the network isup and running.

2. If in "Select Planned Run", select DataManagement in the touch screen,otherwise select Tools4DataManagement from the Main Menu.

3. In the Data Management screen, selectAll, then review the runs. If there are runsthat do not need to be transferred to theTorrent Server (for example test oraborted runs), select the checkbox next tothe run names, then press Delete Sel.

4. If there are runs that you do want totransfer, you may need to wait untilconnectivity is restored for the run totransfer and then autodelete.

Appendix A TroubleshootingInstrument error messages A



Error message: OVERSHOTTARGET PH: W2 pH = n.nnFailed

Auto-pH added more NaOHfrom the Wash 1 Reagent Tubeto the Wash 2 Bottle than wasneeded

1. Re-initialize the instrument using newreagents and a new chip (the chip canthen be used for sequencing).

2. Prepare 50 μL of 100 mM HCl. If you arein the auto-pH screen, press Overshoot.Follow the on-screen prompts to add50 μL of 100 mM HCl to the W2 Solution.This will lower the pH of the W2 Solution.Press Restart to restart auto-pH.

3. If the pH is consistent with the pH of theprevious chip, manually adjust the pH ofthe W2 Solution.

Note: If the Ion Proton™ Systemconsistently overshoots the pH target,add 50 μL of 100 mM NaOH to the Wash 2Bottle prior to installing it on theinstrument. If you continue to experienceovershoot issues even after the additionof the NaOH, contact Technical Supportfor further assistance.

Error message: Please insert achip and press Start

Instrument cannot detect thechip in chip clamp

See recommended action for “Chip is securedin chip clamp, but chip icon indicates no chipdetected“ on page 76.

Error message: Reagent pH:Failed; Reagent pH is displayed

Note: Message displays ontouchscreen after ReagentCheck at end of Initializationprocedure.

One or more reagents are notwithin the target pH

1. Press Restart to restart auto pH andconfirm the measurement.

2. If any reagents fail, replace the chip witha new (unused) one. Insert the chip in thesocket, then press Restart to restart autopH (the new chip can be used forsequencing after initialization completes).

3. If any reagents fail, clean and re-initializethe instrument with fresh reagents and anew chip.




Error message: Reagent pH:Failed; Reagent pH is notdisplayed

Note: Message displays ontouchscreen after ReagentCheck at end of Initializationprocedure.

Chip did not calibrate 1. Remove the chip from the clamp and lookfor damage to the chip, such as hairlinecracks, debris, or a detached flow cell.

• If the chip is damaged, insert a newchip in the chip clamp and engagethe clamp, then press Start to re-runreagent check.

• If no damage is observed, reinsertthe chip in the chip clamp andengage the clamp, then press Startto re-run reagent check.If reagent check fails again, replacethe chip with a new (unused) one,then re-run reagent check (the newchip can be used for sequencingafter initialization completes).

2. If the reagent check continues to fail,contact Technical Support.

Error Message: RemoveConical tubes

• dNTP Reagent Tube seal isnot tight


1. Remove and reinstall each dNTP ReagentTube.

2. Make sure that each tube is securelytightened (finger-tighten), the press OK tore-check pressure.

3. If the error message persists, set updNTPs in new tubes, secure new tubes oninstrument, then press OK.


Appendix A TroubleshootingInstrument error messages A



Error message: There may be ablockage or no NaOH in W1.Please check W1 and run lineclear then try again

The waste lines may be blocked 1. If you are in the auto pH screen, pressLine Clear, otherwise select Tools4AutopH4Line Clear from the Main Menu.

2. Follow the on-screen prompts and usethe syringe provided with the Ion Proton™

System.3. (Optional) To confirm the Line Clear

procedure was successful, select FlowCheck, then confirm that liquid flowsfrom both waste lines.

4. If the flow rates are still not normal,perform Line Clear one more time.

5. If the line(s) remain blocked, contactTechnical Support. Otherwise, ifinitialization was interrupted, restartinitialization from the beginning.

No NaOH added to Wash 1Reagent Tube, so chip does notdetect large enough pHdifference between the NaOHand W2 Solution

1. Remove the Wash 1 Reagent Tube, rinsewith 18 MΩ water, then add 32 µL of 1MNaOH.

2. Replace the Wash 1 Reagent Tube andsecurely tighten.

3. Press Restart to restart auto-pH.

Wash 1 or Wash 2 sipper isloose

1. Loosen the Wash 1 Reagent Tube, tightenthe sipper, then replace the Wash 1 tubeand securely tighten. Tighten the sipperas quickly as possible to minimize the gasflow that occurs when the tube isremoved. (The gas is not harmful to theNaOH solution and is not a hazard.)

2. Loosen the Wash 2 cap and re-tighten thesipper. Tighten the sipper as quickly aspossible to minimize the gas flow thatoccurs when the bottle is removed. (Thegas is not harmful to the W2 Solution andis not a hazard.)

3. Press Restart to re-start the auto-pHprocess.

Error message: UNDERSHOTTARGET PH: W2 pH = n.nnFailed

Auto-pH could not add enoughWash 1 to the Wash 2 beforethe maximum iterations, 10,occurred

1. A blockage may have occurred. Followthe procedure for “Error message: Theremay be a blockage or no NaOH in W1.Please check W1 and run line clear thentry again“ on page 84.

2. Press Restart to re-start auto-pH. If the"Undershot target pH" error appearsagain, replace the chip with a new(unused) chip and restart auto pH.

Note: The new chip can be used forsequencing after initialization completes.




Error message: W2 average notstable. Try reseating/replacingchip

The waste lines may be blocked See first recommended action for “Errormessage: There may be a blockage or noNaOH in W1. Please check W1 and run lineclear then try again“ on page 84.

Reading for W2 Solution is notstabilizing quickly enough

1. Remove the chip from the clamp and lookfor damage to the chip, such as hairlinecracks, debris, or a detached flow cell.

• If the chip is damaged, insert a newchip in the chip clamp and engagethe clamp (the new chip can be usedfor sequencing after initializationcompletes).

• If no damage can be observed,reinsert the chip in the chip clampand engage the clamp.

2. Loosen the Wash 2 Bottle cap and re-tighten the sipper. Tighten the sipper asquickly as possible to minimize the gasflow that occurs when the bottle isremoved. (The gas is not harmful to theW2 Solution and is not a hazard.)

3. Press Restart to re-start auto-pH. If auto-pH fails even after running auto pH with anew chip, contact Technical Support andmanually adjust the pH of the W2Solution.

Error message: W2 out ofrange

• Chip measurements veryunstable

• Chip is damaged

See recommended actions for “Errormessage: W2 average not stable. Tryreseating/replacing chip“ on page 85.

Sample loading or sequencing


Control Ion Sphere™ Particlesare not present in the TestFragment Report section of therun report and librarysequencing is poor

• Poor chip loading

• Control Ion Sphere™

Particles were not addedto the sample

• Chip is damaged

• Instrument failure

1. Confirm that the Control Ion Sphere™

Particles (included in this sequencing kit)were added.

2. If controls were added, contact TechnicalSupport.

Sample results were obtained,but poor or no results forControl Ion Sphere™ Particlesin Test Fragment report

• No Control Ion Sphere™

Particles added to thesample

• Control Ion Sphere™

Particles are past expirydate

Check Control Ion Sphere™ Particles expirydate.

Appendix A TroubleshootingSample loading or sequencing A



Good conversion rate forControl Ion Sphere™ Particles,but poor sample loading andsequencing

Problems with library ortemplate preparation

Verify the quantity and quality of the library andtemplate preparations.

Appendix A TroubleshootingSample loading or sequencingA


ISP Quality Control assay troubleshooting table

The following table contains a troubleshooting information for the unenriched IonSphere™ Quality Control assay on the Qubit® 2.0 or Qubit® 3.0 Fluorometer.

Qubit®

Fluorometerobservation

Ion Proton™ Systemobservation Possible cause Recommended action

<10%Templated

ISPs

• Lower loading

• Lower % enriched

• Lower key signal

• Lower throughput

Too little library input intotemplate preparation

• Increase library input to target 20–25% templated ISPs.or

• Continue with sequencing; expectlower throughput.

>30%Templated

ISPs,

but <70%

Increased number offiltered reads

Too much library input intotemplate preparation

• Decrease library input to target 20–25% templated ISPs.or

• Continue with sequencing; expectlower throughput.

>70%Templated

ISPs

• Increased % primerdimer filtered reads


Adapter dimercontaminating library, morelikely in short amplicon, IonAmpliSeq™or miRNAlibraries

• Check Bioanalyzer® traces foradapter dimer peak (Amplicon libraryor Ion AmpliSeq™ library peak around70 bp; miRNA library peak around60bp).

• Re-purify Agencourt® library usingAMPure® XP Kit clean-up steps asoutlined in the appropriate userguides.

• Low loading

• Low % enriched


• High % filtered reads

Ion Chef™ System underperformance

Troubleshoot with Technical Support or aField Application Scientist.

Appendix A TroubleshootingISP Quality Control assay troubleshooting table A


Supplementary procedures

■ Section: Quality control of Ion PI™ IC 200 Ion Sphere™ Particles . . . . . . . . . . . 89

■ Section: Quality control using the Qubit® 2.0 Fluorometer . . . . . . . . . . . . . . . . . 91

■ Section: Quality control using the Qubit® 3.0 Fluorometer . . . . . . . . . . . . . . . . 104

■ Section: Maintain the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

B


Section: Quality control of Ion PI™ IC 200 IonSphere™ Particles

Quality Control assay

The Ion Sphere™ Quality Control assay on the Qubit® 2.0 or Qubit® 3.0 Fluorometermeasures the fluorescence of template-positive Ion Sphere™ Particles (ISPs) labeledwith two fluorophores: Alexa Fluor® 488 and Alexa Fluor® 647.

• A probe labeled with Alexa Fluor® 488 anneals to primer B sites, or all of the ISPspresent.

• A probe labeled with Alexa Fluor® 647 anneals to primer A sites, or only the ISPswith extended templates.

The ratio of the Alexa Fluor® 647 fluorescence (templated ISPs) to the Alexa Fluor® 488fluorescence (all ISPs present) yields the % templated ISPs.

The following is the schematic of the probes labeled Alexa Fluor® 488 andAlexa Fluor® 647 annealed to an ISP:

Note: The drawing is not to scale.

Quality control using the Qubit® 2.0 or Qubit® 3.0 Fluorometer

The Qubit® 2.0 Fluorometer or the Qubit® 3.0 Fluorometer can be used to perform aquality assessment of unenriched Ion Sphere™ Particles generated for up to base-readsequencing on the Ion Proton™ Sequencer. For details, see “Quality control using theQubit® 2.0 Fluorometer“ on page 91 or “Quality control using the Qubit® 3.0Fluorometer“ on page 104.

Appendix B Supplementary proceduresQuality Control assay B


Quality control using the Guava® easyCyte™ 5 Flow Cytometer

The Guava® easyCyte™ 5 Flow Cytometer can be used for quality assessment ofunenriched and enriched Ion Sphere™ Particles generated for up to 200 base-readsequencing on the Ion Proton™ Sequencer.

• Unenriched samples – Obtain the QC samples from the corresponding LibrarySample Tubes on the Ion PI™ IC Reagents 200 Cartridge (positions A and B).

• Enriched samples – Obtain sample 1 from position E and sample 2 from positionA on the Enrichment Cartridge.

For details, refer to the Ion Sphere™ Particles (ISPs) Quality Assessment Using the Guava®

easyCyte™ 5 Flow Cytometer User Bulletin (Pub. no. 4470082), available on the IonCommunity website:

ioncommunity.lifetechnologies.com

Quality control using the Attune® Cytometer

The Applied Biosystems® Attune® Acoustic Focusing Cytometer can be used forquality assessment of unenriched and enriched Ion Sphere™ Particles generated for upto 200 base-read sequencing on the Ion Proton™ Sequencer.

• Unenriched samples – Obtain the QC samples from the corresponding LibrarySample Tubes on the IC Reagents 200 Cartridge (positions A and B).

• Enriched samples – Obtain sample 1 from position E and sample 2 from positionA on the Enrichment Cartridge.

For details, refer to the Demonstrated Protocol: Ion Sphere™ Particles (ISPs) QualityAssessment using the Applied Biosystems® Attune® Acoustic Focusing Cytometer UserBulletin (Pub. no. 4477181), available on the Ion Community website:

ioncommunity.lifetechnologies.com

IMPORTANT! Life Technologies Demonstrated Protocols have been successfullyverified by Life Technologies for research use. There are no technical specifications forLife Technologies Demonstrated Protocols. Users assume all risk when using theseprotocols, and recognize that support for Life Technologies Demonstrated Protocolsoccurs through community discussion. All customers are encouraged to discuss andcontribute via the Ion Community.

Appendix B Supplementary proceduresQuality control using the Guava® easyCyte™ 5 Flow CytometerB


http://ioncommunity.lifetechnologies.com

http://ioncommunity.lifetechnologies.com

Section: Quality control using the Qubit® 2.0Fluorometer

IMPORTANT! Effective 15 February 2012, the Alexa Fluor® 488 and Alexa Fluor® 647Calibration Standards replace the spectrally similar FAM™ and Cy®5 dyes,respectively, previously used in the kit. To update the existing plugin on the Qubit®

2.0 Fluorometer with the new dye names, install the new plugin file,Ion_PluginV310_AF.qbt.

Both sets of dye-labeled calibration standards can be used in conjunction withfirmware version 3.00 and previous versions of plugin files (ion_plugin.qbt orIon_plugin_AF.qbt).

We highly recommend that you install the new firmware version 3.10 and the new Ionplugin file (Ion_PluginV310_AF) for ease of use as these new versions allow you tooperate the Ion plugin on the Qubit® 2.0 Fluorometer without requiring the USB driveLog to be connected into the device at all times.

The selection options on the user interface of the instrument matches the new AlexaFluor® 488 and Alexa Fluor® 647 Calibration Standards once the new plugin file isinstalled.

Materials required

• Qubit® 2.0 Fluorometer (Cat. no. Q32866) with the V3.10 firmware and the IonSphere™ Quality Control assay

• Qubit® Assay Tubes (Cat. no. Q32856)• PCR tubes, 0.2 mL (Axygen Cat. no. PCR-02-L-C or BioExpress Cat. no. T-3035-1)• Qubit® Easy Calculator Microsoft® Excel® Spreadsheet file containing the

instrument specific Calibration Factor• Ion Sphere™ Quality Control Kit (Cat. no. 4468656)• Unenriched Ion Sphere™ Particles• GeneAmp® PCR System 9700 thermal cycler (Cat. no. N8050200) or equivalent

Appendix B Supplementary proceduresMaterials required B


Upgrade the Qubit® 2.0 Fluorometer firmware and software

This section provides information about upgrading the firmware and the software ofthe Qubit® 2.0 Fluorometer. The following illustration summarizes the upgrade pathfor each firmware version.

Qubit® 2.0 FluorometerFirmware v2.00 or v3.00

Qubit® 2.0 FluorometerFirmware v3.10

To a USB drive, download theprogram and upgrade files

(Ion_PluginV310_AF.qbt, V3.10Qubit_FW_MainCPU.bin, and

V3.10 Qubit_FW_UsbHost.bin).

To a USB drive, download theprogram file

(Ion_PluginV310_AF.qbt).

q q

Power on the Qubit® 2.0Fluorometer and connect the

USB drive.

Power on the Qubit® 2.0Fluorometer and connect the

USB drive.

q q

Touch V2.00 or V3.00.When prompted, touch Yes to

upload the Ion Sphere™ QualityControl Assay for V3.10.

q

Touch Update.

q

When prompted, touch Yes toupload the Ion Sphere™ Quality

Control Assay for V3.10.

Appendix B Supplementary proceduresUpgrade the Qubit® 2.0 Fluorometer firmware and softwareB


Upgrade for firmware V2.00 or V3.00

Note: The previous program files (.qbt) are not compatible with the new V3.10firmware. The new program file (Ion_PluginV310_AF.qbt) is not compatible withV3.00 firmware.

• Qubit® 2.0 Fluorometer (Part no. Q32866)• USB drive (included with Qubit® 2.0 Fluorometer)• Program (.qbt) and upgrade (.bin) files:

– Ion_PluginV310_AF.qbt– V3.10 Qubit_FW_MainCPU.bin– V3.10 Qubit_FW_UsbHost.bin

We recommend that you use a Windows® OS-based computer to download andtransfer the Qubit® 2.0 Fluorometer files to the USB drive.

Note: Do not use a Macintosh® computer to transfer the files to the USB drive if theserial number of your Qubit® 2.0 Fluorometer is lower than 1104004846.

We also recommend that you use the USB drive provided with Qubit® 2.0Fluorometer for file transfer.

Other compatible USB drives may also be used. Visit:http://ioncommunity.lifetechnologies.com/community/products/pgm/ to see a list ofapproved USB drives. Logging into the Ion Community is required.

1. Download the program file (.qbt) and two upgrade files (.bin) fromhttp://ioncommunity.lifetechnologies.com/community/products/pgm/ onto aWindows®-based PC desktop. Logging into the Ion Community is required.

2. Remove the existing program file (.qbt) and two upgrade files (.bin) from theUSB drive, if any.

3. Transfer the three downloaded files to the USB drive provided with the Qubit®

2.0 Fluorometer (recommended), or another compatible USB drive.

Note: Files on the USB drive must be in the root directory and cannot be in afolder.

1. Power on the Qubit® 2.0 Fluorometer by plugging in the unit.

2. Insert the USB drive, containing the program (.qbt) and upgrade (.bin) files, intothe USB port on the instrument.

3. In the upper-right corner of the main menu, touch V2.00, or V3.00 depending onthe version.

4. If the image of the USB drive in the update screen displays a green dot, touchUpdate to upgrade the instrument firmware.The firmware update lasts approximately 2 minutes, during which the Qubit® 2.0Fluorometer screen flashes:

Materialsrequired forupdate

Guidelines fordownloading files

Upgrade thefirmware from theprevious versions

Appendix B Supplementary proceduresUpgrade for firmware V2.00 or V3.00 B


http://ioncommunity.lifetechnologies.com/community/products/pgm/user_guides_and_bulletins




Note: If the image displays a red dot, the Qubit® 2.0 Fluorometer cannot detectthe USB drive. Ensure the USB drive is in place securely. If the problem persists,remove the USB drive and reinsert.

5. When the Qubit® 2.0 Fluorometer displays the main screen, confirm that V3.10 isdisplayed (in the upper-right corner), which confirms that the upgrade wassuccessful.After the firmware has been updated, the following screen will be displayed.Confirm that V3.10 is displayed in the upper-right corner. Touch Yes topermanently upload the Ion Sphere™ QC program to the instrument:

Appendix B Supplementary proceduresUpgrade for firmware V2.00 or V3.00B


6. Confirm the Ion Sphere™ QC program (.qbt) file is functional by checking thefollowing screens.

a. In the main menu, the Ion selection option is present:

b. After touching Ion ( ), AF 488 ( ) and AF 647 ( ) selectionoptions appear on the screen:

Note: Touching AF 488 or AF 647 enters the respective measurementchannels.

7. Proceed to complete the instruction in “Calculate the Qubit® 2.0 FluorometerCalibration Factor“ on page 97.

Upgrade for firmware V3.10

If your Qubit® 2.0 Fluorometer is equipped with V3.10 firmware, you only need toupload the Ion Sphere™ Quality Control assay into your fluorometer:

Firmware version

IMPORTANT! Do not re-upgrade the firmware if the instrument is shipped withV3.10 version.

Appendix B Supplementary proceduresUpgrade for firmware V3.10 B


To upload the Ion Sphere™ Quality Control assay into your fluorometer, use thefollowing steps:

1. Download the new Ion_PluginV310_AF.qbt file (from:http://ioncommunity.lifetechnologies.com/community/products/pgm/) to your USBdrive to activate the application to accept the new names. Logging into the IonCommunity is required.

Note: We recommend that you use the USB drive provided with Qubit® 2.0Fluorometer for file transfer; however, other compatible USB drives may also beused. Visit:http://ioncommunity.lifetechnologies.com/community/products/pgm/ to see a list of approved USB drives. Logging into the Ion Community isrequired.

2. With the USB drive (containing the Ion_PluginV310_AF.qbt file) inserted into theQubit® 2.0 Fluorometer, power-cycle the instrument by unplugging andplugging it back in. Touch Yes to permanently upload the file to the instrument:

3. Confirm the Ion Sphere™ QC program (.qbt) file is functional by checking thefollowing screens:

a. In the main menu, the Ion selection option is present.

Appendix B Supplementary proceduresUpgrade for firmware V3.10B






b. After touching Ion ( ), AF 488 ( ) and AF 647 ( ) selectionoptions appear on the screen:

Note: Touching AF 488 or AF 647 enters the respective measurementchannels.

4. Proceed to “Calculate the Qubit® 2.0 Fluorometer Calibration Factor“.

Calculate the Qubit® 2.0 Fluorometer Calibration Factor

This section describes the procedure to determine the Qubit® 2.0 Fluorometerinstrument-specific Calibration Factor.

IMPORTANT! You must upgrade the Qubit® 2.0 Fluorometer firmware and softwareprior to performing the following procedure. See “Upgrade the Qubit® 2.0Fluorometer firmware and software“ on page 92 for more information.

Each Qubit® 2.0 Fluorometer has a unique Calibration Factor that must be calculatedand applied to all Percent Templated ISPs calculations.

Note: It is only necessary to calculate the Calibration Factor once for a particularinstrument, unless a problem is suspected.

• Qubit® Easy Calculator Microsoft® Excel® Spreadsheet (Logging into the IonCommunity is required.)

• Qubit® 2.0 Fluorometer (Part no. Q32866) with V3.10 firmware• USB drive containing the “.qbt” file• Qubit® Assay Tubes (Part no. Q32856)• Ion Sphere™ Quality Control Kit (Cat. no. 4468656)

Download the Qubit® Easy Calculator Microsoft® Excel® Spreadsheet file from: http://ioncommunity.lifetechnologies.com/community/products/pgm/ (Logging into theIon Community is required), and save the file to the computer used for Qubit® 2.0Fluorometer data analysis.

Materialsrequired

Download theQubit® EasyCalculator

Appendix B Supplementary proceduresCalculate the Qubit® 2.0 Fluorometer Calibration Factor B




1. From the Ion Sphere™ Quality Control Kit, thaw the Alexa Fluor® 488 andAlexa Fluor® 647 Calibration Standard reagents.

Note: Both the Alexa Fluor® 488 and Alexa Fluor® 647 molecules arephotosensitive, so avoid exposure to light for long periods of time and directsunlight.

2. Vortex well to mix and pulse-spin the tube to remove any liquid trapped in thecap.

3. Transfer 200 µL of each standard into two separate Qubit® assay tubes. Pulse-spin to bring all the liquid to the bottom of the tube.

IMPORTANT! Prior to using the Qubit® 2.0 Fluorometer, ensure that the instrument isrunning Firmware V3.10, and the Ion_PluginV310_AF.qbt file has been permanentlyuploaded to the instrument. See section “Upgrade the Qubit® 2.0 Fluorometerfirmware and software“ on page 92 for more information about managing Qubit® 2.0Fluorometer firmware versions.

1. Touch Ion to access Alexa Fluor® 488 and Alexa Fluor® 647 measurementoptions.

2. Touch AF 488 and insert the Alexa Fluor® 488 Calibration Standard reagent intothe Qubit® 2.0 Fluorometer, close the lid, and touch Read.

Note: The lettering on the Read ( ) selection option changes from white tored when reading a sample. The lettering changes back to white when thereading of the sample is finished in approximately 5 seconds.

3. Record the RFU value and remove sample from Qubit® 2.0 Fluorometer.

4. Touch Home, touch Ion, and then touch AF 647. Insert the Alexa Fluor® 647Calibration Standard into the Qubit® 2.0 Fluorometer, close the lid, and touchRead.


5. Record the RFU value and remove sample from Qubit® 2.0 Fluorometer.

1. In the Qubit® Easy Calculator, enter each recorded RFU value in theappropriately labeled green cell to display the Calibration Factor specific for theQubit® 2.0 Fluorometer.

2. Save a copy of the Qubit® Easy Calculator containing the Calibration Factor foruse as a template for future Percent Templated ISPs calculations:

Note: Affix a sticker with the instrument-specific Calibration Factor to theQubit® 2.0 Fluorometer.

Calibrationstandardpreparation

Calibrationstandardmeasurement

Calibration Factorcalculation

Appendix B Supplementary proceduresCalculate the Qubit® 2.0 Fluorometer Calibration FactorB


IMPORTANT! For each Qubit® 2.0 Fluorometer used, save a separate Qubit®

Easy Calculator Microsoft® Excel® Spreadsheet file containing the CalibrationFactor specifically calculated for that particular instrument.



Measure the templated unenriched sample

This section describes the procedure for determining the percent templated ISPs forunenriched Ion Sphere™ Particles.

1. From the Ion Sphere™ Quality Control Kit, thaw the Ion Probes tube, AnnealingBuffer, and Quality Control Wash Buffer.

2. Centrifuge the samples taken from Positions A and B on the IC Reagents 200Cartridge at maximum speed for 3 minutes to reduce the total volume persample to approximately 10 µL.

D

A

B

C

00012647



Note: If you want use both Guava® flow cytometry and Qubit® fluorometry forquality assessment, remove 1 µL of the sample prior to centrifugation for Guava®

analysis before processing the remainder for Qubit® analysis.

Note: If you are assessing quality after completion of the Ion Chef™ run, youmay remove the Library Sample Tubes from the reagents cartridge andcentrifuge the QC samples in these tubes.

3. Pipet each sample up and down to mix and then transfer each sample to a newlabeled 0.2-mL PCR tube.

4. Add 10 µL Annealing Buffer and 1 µL Ion Probes directly to each 0.2-mL PCRtube containing the ISPs, then mix well by pipetting up and down.

Note: If processing multiple samples, generate an Ion Probe Master Mix asfollows:(10 µL Annealing Buffer × # samples) + (1 µL Ion Probes × # samples) = totalvolume required

5. Load the tube(s) into a thermal cycler, then perform the following protocol toanneal the Ion Probes:

Stage Temperature Time

Hold 95ºC 2 min

Hold 37ºC 2 min

6. Remove unbound probes by washing the sample(s) three times with 200 µL ofQuality Control Wash Buffer.

a. Add 200 µL of Quality Control Wash Buffer to the 0.2-mL tube(s).

Samplepreparation

Appendix B Supplementary proceduresMeasure the templated unenriched sampleB


b. Vortex properly to mix and centrifuge at maximum speed for 3 minutes.

c. Being careful not to disturb the pelleted ISPs, remove the supernatant andleave behind 10 µL.

Note: Compare to a 10 µL standard for reference.

d. Repeat substep a through substep c two times for a total of three QualityControl Wash Buffer washes.

7. After the final wash, add 190 µL of Quality Control Wash Buffer for a totalvolume of 200 µL, mix by pipetting up and down five times and transfer theentire sample to a Qubit® assay tube.

IMPORTANT! Ensure that you measure the volumes accurately.

8. To generate a negative control, add 200 µL of Quality Control Wash Buffer to afresh Qubit® assay tube.

9. Read the sample(s) as described below.

IMPORTANT! Prior to using the Qubit® 2.0 Fluorometer, ensure that the instrument isrunning Firmware V3.10, and the Ion_PluginV310_AF.qbt file has been permanentlyuploaded to the instrument. See section “Upgrade the Qubit® 2.0 Fluorometerfirmware and software“ on page 92 for more information about managing Qubit® 2.0Fluorometer firmware versions.

1. Power on the Qubit® 2.0 Fluorometer.

2. Touch Ion to access Alexa Fluor® 488 and Alexa Fluor® 647 measurementoptions.

3. Touch AF 488 and insert the sample into the Qubit® 2.0 Fluorometer, close thelid, and touch Read.


Note: If more than one sample is being processed, all samples can be read withthe AF 488 setting before moving on to the AF 647 setting.

4. Record the value.

Note: The data retained on the Qubit® 2.0 Fluorometer can be transferred to aUSB drive. See the “(Optional) Data Transfer to USB Drive“ on page 102 fordetails. If more than one sample is being processed, all samples can be read withthe AF 488 setting before moving on to the AF 647 setting.

5. Touch Home, touch Ion, and then touch AF 647. Insert the sample into theQubit® 2.0 Fluorometer, close the lid, and touch Read.


Samplemeasurement

Appendix B Supplementary proceduresMeasure the templated unenriched sample B



IMPORTANT! Ensure that you read the negative control (Quality Control WashBuffer only) in both the Alexa Fluor® 488 and Alexa Fluor® 647 settings andrecord the RFU values.

1. Ensure that the USB drive is inserted in the instrument.

2. In the main menu, touch Data (at the bottom-right corner of the screen).

3. In the data screen, touch USB drive, then wait for the instrument to downloadthe data to the USB drive:

Note: The download creates a “.csv” file that can be opened on your computerusing any spreadsheet software, such as Microsoft® Excel® software.

1. Open the saved Qubit® Easy Calculator Microsoft® Excel® Spreadsheet filecontaining the Calibration Factor specifically calculated for the Qubit®

Fluorometer used.

2. Enter the raw RFU values from Alexa Fluor® 488 and Alexa Fluor® 647Calibration Standards measurements in the appropriate fields for both the ISPscontaining samples (red cells) and negative control sample (purple cells).

IMPORTANT! The Alexa Fluor® 488 value must be >100 counts to produce avalid % Templated ISPs value. If the Alexa Fluor® 488 value is <100 counts, seethe “ISP Quality Control assay troubleshooting table“ on page 87.

Fluorophore Acceptable RFU range

Alexa Fluor® 488 >100 counts; no upper limitSamples with <100 counts usually correlate with no or veryfew ISPs in the assay.

Alexa Fluor® 647 Any value, with the condition that the Alexa Fluor® 488 RFUvalue is >100 counts.

3. In the appropriate field (blue cells), enter the lot-specific conversion factor forunenriched Ion Sphere™ Particles, available at: http://ioncommunity.lifetechnologies.com/docs/DOC-9093. (Log in is required.)

(Optional) DataTransfer to USBDrive

Templated ISPevaluation



http://ioncommunity.lifetechnologies.com/docs/DOC-9093


4. The Percent Templated ISPs calculates automatically and is displayed for eachsample:

Acceptance criteria for unenriched Ion Sphere™ Particles

The optimal amount of library corresponds to the library dilution point that givesPercent Templated ISPs between 10–30%.

Samples that fall within the recommended range generally produce the most data;however, samples that fall outside of the recommended range can still meet thethroughput specifications on the Ion chips.

The recommended optimal range is not intended to be a pass/fail criteria. The rangeprovides guidance for the quality of the sample.

Note: If the results are outside the desired Percent Templated ISPs range, thenincrease or decrease the library input appropriately. See the “ISP Quality Controlassay troubleshooting table“ on page 87 for more information.

Percent Templated ISPs Description

<10% Sample contains an insufficient number of templated ISPsto achieve optimal loading density on the Ion Chip.

10–30% Optimal amount of library.

>30% Sample will yield multi-templated ISPs (mixed reads).

Appendix B Supplementary proceduresAcceptance criteria for unenriched Ion Sphere™ Particles B


Section: Quality control using the Qubit® 3.0Fluorometer

The Qubit® 3.0 Fluorometer features a pre-loaded Ion Sphere™ quality control assayfor determining the percentage of templated ISPs. As with the Qubit® 2.0 Fluorometer,a unique instrument-specific Calibration Factor must be calculated and applied to allpercent templated ISP calculations.

Materials required

• Qubit® 3.0 Fluorometer (Cat. no. Q33216)• Qubit® Assay Tubes (Cat. no. Q32856)• PCR tubes, 0.2 mL (Axygen Cat. no. PCR-02-L-C or BioExpress Cat. no. T-3035-1)• Qubit® Easy Calculator Microsoft® Excel® Spreadsheet file containing the

instrument-specific Calibration Factor• Ion Sphere™ Quality Control Kit (Cat. no. 4468656)• Unenriched Ion Sphere™ Particles• GeneAmp® PCR System 9700 thermal cycler (Cat. no. N8050200) or equivalent

Calculate the Qubit® 3.0 Fluorometer Calibration Factor

This section describes the procedure to determine the Qubit® 3.0 Fluorometerinstrument-specific Calibration Factor.

Note: It is only necessary to calculate the Calibration Factor once for a particularinstrument, unless a problem is suspected.

Download the Qubit® Easy Calculator Microsoft® Excel® Spreadsheet file from: http://ioncommunity.lifetechnologies.com/community/products/pgm/ (Logging into theIon Community is required), and save the file to the computer used for Qubit® 3.0Fluorometer data analysis.

1. From the Ion Sphere™ Quality Control Kit, thaw the Alexa Fluor® 488 andAlexa Fluor® 647 Calibration Standard reagents.

Note: Both the Alexa Fluor® 488 and Alexa Fluor® 647 molecules arephotosensitive, so avoid exposure to light for long periods of time and directsunlight.

2. Vortex well to mix and pulse-spin the tube to remove any liquid trapped in thecap.

3. Transfer 200 µL of each standard into two separate Qubit® assay tubes. Pulse-spin to bring all the liquid to the bottom of the tube.

Download theQubit® EasyCalculator

Calibrationstandardpreparation

Appendix B Supplementary proceduresMaterials requiredB





2. Touch Ion Sphere on the Qubit® 3.0 Fluorometer home screen open the IonSphere™ Assay. Touch AF 488.

3. Insert the Alexa Fluor® 488 Calibration Standard reagent into the Qubit® 3.0Fluorometer, close the lid, and touch Read tube ( ).

4. Record the RFU value, remove the assay tube from the Qubit® 3.0 Fluorometerand touch the Home icon in the upper left corner of the screen.

Calibrationstandardmeasurement



5. On the Home screen, touch Ion Sphere, and then touch AF 647. Insert theAlexa Fluor® 647 Calibration Standard into the Qubit® 3.0 Fluorometer, close thelid, and touch Read tube ( ).

6. Record the RFU value, remove the assay tube from the Qubit® 3.0 Fluorometer.Touch Data ( ).

7. Touch Export ( ) to export data to a USB storage drive or to a USB-connected computer. Touch Done ( ) to return to the Home screen.

Appendix B Supplementary proceduresCalculate the Qubit® 3.0 Fluorometer Calibration FactorB


1. In the Qubit® Easy Calculator, enter each recorded RFU value in theappropriately labeled green cell to display the Calibration Factor specific for theQubit® 3.0 Fluorometer.

2. Save a copy of the Qubit® Easy Calculator containing the Calibration Factor foruse as a template for future Percent Templated ISPs calculations:

Note: Affix a sticker with the instrument-specific Calibration Factor to theQubit® 3.0 Fluorometer.

IMPORTANT! For each Qubit® 3.0 Fluorometer used, save a separate Qubit®

Easy Calculator Microsoft® Excel® Spreadsheet file containing the CalibrationFactor specifically calculated for that particular instrument.

Calibration Factorcalculation



Measure the templated unenriched sample

This section describes the procedure for determining the percent templated ISPs forunenriched Ion Sphere™ Particles.

1. From the Ion Sphere™ Quality Control Kit, thaw the Ion Probes tube, AnnealingBuffer, and Quality Control Wash Buffer.

2. Centrifuge the samples taken from Positions A and B on the IC Reagents 200Cartridge at maximum speed for 3 minutes to reduce the total volume persample to approximately 10 µL.

D

A

B

C

00012647



Note: If you want use both Guava® flow cytometry and Qubit® fluorometry forquality assessment, remove 1 µL of the sample prior to centrifugation for Guava®

analysis before processing the remainder for Qubit® analysis.

Note: If you are assessing quality after completion of the Ion Chef™ run, youmay remove the Library Sample Tubes from the reagents cartridge andcentrifuge the QC samples in these tubes.

3. Pipet each sample up and down to mix and then transfer each sample to a newlabeled 0.2-mL PCR tube.

4. Add 10 µL Annealing Buffer and 1 µL Ion Probes directly to each 0.2-mL PCRtube containing the ISPs, then mix well by pipetting up and down.

Note: If processing multiple samples, generate an Ion Probe Master Mix asfollows:(10 µL Annealing Buffer × # samples) + (1 µL Ion Probes × # samples) = totalvolume required

5. Load the tube(s) into a thermal cycler, then perform the following protocol toanneal the Ion Probes:

Stage Temperature Time

Hold 95ºC 2 min

Hold 37ºC 2 min

6. Remove unbound probes by washing the sample(s) three times with 200 µL ofQuality Control Wash Buffer.

a. Add 200 µL of Quality Control Wash Buffer to the 0.2-mL tube(s).

Samplepreparation



b. Vortex properly to mix and centrifuge at maximum speed for 3 minutes.

c. Being careful not to disturb the pelleted ISPs, remove the supernatant andleave behind 10 µL.

Note: Compare to a 10 µL standard for reference.

d. Repeat substep a through substep c two times for a total of three QualityControl Wash Buffer washes.

7. After the final wash, add 190 µL of Quality Control Wash Buffer for a totalvolume of 200 µL, mix by pipetting up and down five times and transfer theentire sample to a Qubit® assay tube.

IMPORTANT! Ensure that you measure the volumes accurately.

8. To generate a negative control, add 200 µL of Quality Control Wash Buffer to afresh Qubit® assay tube.

9. Read the sample(s) as described below.


2. Touch Ion Sphere to access Alexa Fluor® 488 and Alexa Fluor® 647 measurementoptions.

3. Touch AF 488 and insert the sample into the Qubit® 3.0 Fluorometer, close thelid, and touch Read tube.

Note: If more than one sample is being processed, all samples can be read withthe AF 488 setting before moving on to the AF 647 setting.


Note: The data retained on the Qubit® 3.0 Fluorometer can be transferred to aUSB drive. See the “(Optional) Data transfer to USB drive or computer“ fordetails. If more than one sample is being processed, all samples can be read withthe AF 488 setting before moving on to the AF 647 setting.

5. Touch Home, touch Ion Sphere, and then touch AF 647. Insert the sample intothe Qubit® 3.0 Fluorometer, close the lid, and touch Read tube.


IMPORTANT! Ensure that you read the negative control (Quality Control WashBuffer only) in both the Alexa Fluor® 488 and Alexa Fluor® 647 settings andrecord the RFU values.

Samplemeasurement

Appendix B Supplementary proceduresMeasure the templated unenriched sample B


1. Ensure that the USB drive is inserted in the instrument, or a computer isconnected by USB cable.

2. On the Home screen, touch Data (at the bottom-left of the screen).

3. On the Data screen, touch Export, then wait for the instrument to download thedata to the USB drive or computer.

Note: The download creates a “.csv” file that can be opened on your computerusing any spreadsheet software, such as Microsoft® Excel® software.

1. Open the saved Qubit® Easy Calculator Microsoft® Excel® Spreadsheet filecontaining the Calibration Factor specifically calculated for the Qubit®

Fluorometer used.

2. Enter the raw RFU values from Alexa Fluor® 488 and Alexa Fluor® 647Calibration Standards measurements in the appropriate fields for both the ISPscontaining samples (red cells) and negative control sample (purple cells).

IMPORTANT! The Alexa Fluor® 488 value must be >100 counts to produce avalid % Templated ISPs value. If the Alexa Fluor® 488 value is <100 counts, seethe “ISP Quality Control assay troubleshooting table“ on page 87.

Fluorophore Acceptable RFU range

Alexa Fluor® 488 >100 counts; no upper limitSamples with <100 counts usually correlate with no or veryfew ISPs in the assay.

Alexa Fluor® 647 Any value, with the condition that the Alexa Fluor® 488 RFUvalue is >100 counts.

3. In the appropriate field (blue cells), enter the lot-specific conversion factor forunenriched Ion Sphere™ Particles, available at: http://ioncommunity.lifetechnologies.com/docs/DOC-9093. (Log in is required.)

(Optional) Datatransfer to USBdrive or computer

Templated ISPevaluation





4. The Percent Templated ISPs calculates automatically and is displayed for eachsample:

Acceptance criteria for unenriched Ion Sphere™ Particles

The optimal amount of library corresponds to the library dilution point that givesPercent Templated ISPs between 10–30%.

Samples that fall within the recommended range generally produce the most data;however, samples that fall outside of the recommended range can still meet thethroughput specifications on the Ion chips.

The recommended optimal range is not intended to be a pass/fail criteria. The rangeprovides guidance for the quality of the sample.

Note: If the results are outside the desired Percent Templated ISPs range, thenincrease or decrease the library input appropriately. See the “ISP Quality Controlassay troubleshooting table“ on page 87 for more information.

Percent Templated ISPs Description

<10% Sample contains an insufficient number of templated ISPsto achieve optimal loading density on the Ion Chip.

10–30% Optimal amount of library.

>30% Sample will yield multi-templated ISPs (mixed reads).

Appendix B Supplementary proceduresAcceptance criteria for unenriched Ion Sphere™ Particles B


Section: Maintain the Ion Chef™ SystemThe Ion Chef™ System supports the user-performed maintenance procedures found inthis section.

Install a firmware update

To ensure proper operation of the Ion Chef™ Instrument, we recommend periodicallyconfirming that your instrument is running the most current firmware. Occasionally,Life Technologies may release updates to the instrument firmware, which can includeimportant changes to the Ion Chef™ System operation. To ensure that your instrumentis running the most current firmware, check the firmware version as explained below.

1. On the Ion Chef™ Instrument touchscreen, touch Settings.

2. On the Settings screen, touch Check for updates.

3. On the Software Update screen, touch Release to search the server for updatesto the Ion Chef™ Instrument firmware.

Note: Users are also notified of the availability of firmware updates on theNotifications screen.

4. Select the available component update, touch Update, then wait for theinstrument to complete the update.

Note: If the Ion Chef™ Instrument is operating on an isolated network andcannot connect to the Life Technologies website, you must transfer the firmwareupdate manually. To perform a manual update, transfer the firmware files to aUSB drive and insert the drive into the USB port at the rear of the instrument.Touch USB on the Software Update screen, select from the list of availablesoftware components, then touch Update.

When finished, the Ion Chef™ Instrument displays the update status.

5. Power off and on the Ion Chef™ Instrument to complete the update.

IMPORTANT! If you are updating the firmware manually, remove the USBdrive before powering on the Ion Chef™ Instrument.

Check the IonChef™ Instrumentfirmware

Appendix B Supplementary proceduresInstall a firmware updateB


Change the instrument name

The following procedure describes how to change the name used to identify the IonChef™ Instrument both on the network and in the data that it generates.

1. In the Ion Chef™ Instrument home screen, touch Settings.

2. In the Settings screen, touch Instrument settings.

3. In the Instrument settings screen, touch Set Instrument Name. Touch the namefield and enter a new instrument name using the keypad that appears, thentouch Save.

Note: Use only alphanumeric characters. Do not use special characters or spacesin the name.

4. In the next screen, touch Save again. Power off and on the Ion Chef™ Instrumentto effect the name change.

Appendix B Supplementary proceduresChange the instrument name B


Ion Chef™ and Torrent Servernetwork setup

■ Networking overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

■ Ion Chef™ Networking Configuration Options . . . . . . . . . . . . . . . . . . . . . . . . . . 118

■ Editing the Torrent Server firewall tables to allow the LAN port IP . . . . . . . . 125

■ Enable Planned Run sharing among Torrent Servers . . . . . . . . . . . . . . . . . . . . . 127

■ Transfer a Planned Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

■ Undo a Planned Run transfer (administrator) . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

■ Undo a Planned Run transfer (user) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

Networking overview

If you are preparing a laboratory for use with new Ion instruments, refer to therespective Site Preparation and Safety guides. If you currently have an Ion Chef™

instrument configured in your laboratory to communicate to more than one TorrentServer, the release of Torrent Suite™ v4.4 Software will require the reconfiguration ofthe Ion Chef™ instrument to communicate with only one primary Torrent Server.After upgrading to Torrent Suite v4.4, follow the guidelines in this document toconfigure the instrument to communicate with a primary Torrent Server. From thatTorrent Server, the new Planned Run sharing option can be used to target additionalTorrent Servers if needed.

If you need to connect a Torrent Server to the Network for the first time, see theTorrent Server networking requirements in the site prep and safety guide for therespective sequencer.

This guide describes how to:

• Set up the Ion Chef™ instrument to talk to the Torrent Server on a LAN.• Enable Planned Run sharing.

Note: If you added an Ion Chef™ instrument to your LAN and your Torrent Servershave Torrent Suite™ software version 4.2, upgrading your Torrent Servers to softwareversion 4.4 will require Planned Run sharing to see more than one Torrent Server. TheIon Chef™ instrument is now programmed to see only one primary Torrent Server(previously, it could see two). Configuring Planned Run sharing on Torrent Serverswill allow your Ion Chef™ instrument to share planned runs with multiple otherTorrent Servers (and ultimately, sequencers). This requires the primary Torrent Serverto be chosen as the Ion Chef™server and it will be the starting point (origin server).Once chosen and configured, this Torrent Server can be programmed to sharePlanned Runs with multiple other Torrent Servers, hence allowing an Ion Chef™

instrument to share Planned Runs with multiple servers.

C


The instrument is factory-configured for IPv4 TCP/IP communication and includes afast Ethernet adapter (1000 Mbps) with a RJ45-type connector for integrating thedevice into a local area network (LAN). If the instrument will be connected to a LAN,an active network port must be available/assigned in place before the scheduledinstallation date.

If you are activating a newly installed LAN port for your Ion Chef™ instrument,please ensure the LAN has been configured to permit HTTP-443, SSH-22, andFTP-20/21 traffic. For additional information, please refer to the NetworkingRequirements section of the Ion Chef™ Site Preparation Guide (Pub. no. MAN0007956).

The Torrent Server is an integral part of the system and includes a quad-port gigabitNIC for direct communication with the network or instruments such as Ion Chef™,PGM™, or Ion Proton™ instruments. When the Torrent Server is connected to thenetwork, Torrent Servers that are on the same software versions and subnet cantransfer planned runs from one Torrent Server to another.

Consult a network administrator.

We recommend that you consult a network administrator before connecting the IonChef™ instrument to your laboratory network. Please refer to the networkingrequirements in the Ion Chef™ System Site Preparation Guide.

Collect the required network information.

q

Connect the Ion Chef™ instrument to the configured LAN port.q

Configure the Ion Chef™ instrument with the required information.q

Monitor the Ion Chef™ instrument to test the network connection.

Note: This guide provides a basic description of how to integrate the Torrent Serverand the Ion Chef™ instruments into several possible network architectures. Becauseyour network may contain advanced features, such as firewall or network domains,we recommend that you consult a network administrator before connecting IonChef™instrument to your laboratory network.

Network examples include:• Direct connection• Static IP (indirect connection)• DHCP IP (indirect connection)

Obtain the following information from your network administrator:• Network policy for obtaining IP addresses (DHCP IP or static IP).• If the network requires static IP addresses, obtain the static IP address, subnet

mask, gateway address, and DNS server name for the LAN port.• For the Planned Run sharing and FTP functionality to be successful, the subnet

must be the same on all Torrent Servers and Ion Chef™ instruments on the LAN.

About the IonChef™ instrument

About the TorrentServer

Networkingguidelines andbest practices

Network setupworkflow

Network setupexamples

Collect therequired networkinformation

Appendix C Ion Chef™ and Torrent Server network setupNetworking overview C


Ethernet cable with RJ45 connectors (CAT6 Ethernet cable for a 1000Mbit/s networkconnection or a CAT5 for 100Mbit/s connection).

See Ion Chef™ System Site Preparation Guide (Pub. no. MAN0007956).

DHCP IP

If using DHCP for the LAN port, please make sure the port is set to auto-populatewhen the Ion Chef™ instrument is connected and ensure that all the belowrequirements are met.

Static IP

If the network requires static IP addresses, obtain the static IP address, subnet mask,gateway address, and DNS server name for the LAN port.

For planned run sharing and FTP functionality to be successful, the subnet must bethe same on all Torrent Servers and Ion Chef™ instruments on the LAN.

Materialsrequired

Generalrequirements

Appendix C Ion Chef™ and Torrent Server network setupNetworking overviewC


Direct connection

To configure the direct connection between an Ion Chef™ instrument and a TorrentServer, you will need a Category 6 cable of sufficient length.

Another direct connection configuration option consists of one Ion Chef™ instrument,one Torrent Server and two Ion PGM™ sequencers. However, this configuration is notrecommended due to increased analysis times.

WARNING! The direct configuration shown below is known to result inlengthy analysis times.

Appendix C Ion Chef™ and Torrent Server network setupNetworking overview C


Ion Chef™ Networking Configuration Options

With the Torrent Suite™ software v4.4 release, the Ion Chef™ software will onlyconnect to one Torrent Server. From that server, Planned Run sharing can beconfigured to access additional servers. When the Ion Chef™ is connected to a TorrentServer, an icon on the lower left side of the screen appears.

Connect the Ion Chef™ Instrument to the Torrent Server in one of three ways: Direct,Static IP (non-direct) or DHCP IP (non-direct). See respective instructions below.

Option Description

Directconnection

1. Verify Torrent Server connection on the Ion Chef™ Instrument.

2. The Ion Chef™ Instrument can plug directly into one of the availableports on the back of the Torrent Server. The ports have the address192.168.201 (202, 203).1 according to which port you are connectedto.

Appendix C Ion Chef™ and Torrent Server network setupIon Chef™ Networking Configuration OptionsC


Option Description

3. Configure the Ion Chef™ Instrument.

4. Go to Settings > Torrent Server.

5. Verify the Torrent Server settings are correct. The Torrent BrowserURL will be the IP address of the port you are connected to on theback of the Torrent Server (192.168.201(or .202, or .203).1 and ensurethat the browser password is correct.

6. The FTP address is now directly linked to the above address .

7. Set Password to customer's ionadmin account password.

Appendix C Ion Chef™ and Torrent Server network setupIon Chef™ Networking Configuration Options C


Option Description

8. Enter ionguest as the FTP password.

Static IP 1. If you are going to connect to the Torrent Server through thecompany LAN configured as a Static IP, then you will need toconfigure the customer provided Static IP, Gateway, Netmask, andDNS Server Names. (Only one DNS server is required for the IonChef™ Instrument.)

2. Go toSettings4Instrument Settings4Network4 Configuration.

3. Select the Static IP radio button.

4. Enter the addresses and press Done.

DHCP IP(non-direct)

1. To connect to the Torrent Server through the company LAN, you willneed to target ServerPortIP (Eth1). With LAN set to DHCP, the IP,Gateway, Netmask, and DNS Name Servers should populateautomatically.

2. Go toSettings4Instrument Settings4Network Configuration4.

3. Verify the DHCP radio button is selected.



Option Description

4. Confirm the information is correct. Press Done.

To target the Torrent Server IP address from the Ion Chef™user interface, you will firstlook up the Torrent Server IP address and then enter it into the Ion Chef™userinterface as the Torrent Browser target and the Update Target.

1. Log into Torrent Browser as the admin.

2. Click the Gear button and select Configure.

Target the TorrentServer IP Address



3. Scroll down to the Database Administration section and click the AdminInterface link.



4. The Site Administration screen appears. Scroll to the bottom and click ViewNetwork Settings.



5. Note (copy or write down) the Torrent Server IP address displayed on theNetwork Settings screen.

6. Go back to the Ion Chef™ user interface and tap on the Torrent Browser IPaddress. This opens the Manage Connections window.



7. Enter the server IP address under Server and repeat for Update Site. Click Save.

Editing the Torrent Server firewall tables to allow the LAN port IP

In addition, when connecting through a LAN, the Torrent Server firewall settings willneed to be modified for both DHCP and Static LAN connections.

Note: While directly connected, instruments are on the 192.168.20x.x subnet fromwhich all traffic is enabled, so this is not required.

Any exception would mean that editing the local firewall tables to include the LAN IPis required. Also, if the Ion Chef™ instrument is getting a DHCP address from yournetwork, you will need to create an exception for the entire DHCP subnet.

1. On the Torrent Server, edit the /etc/iptables.custom file and add a firewall rule toallow all traffic from Ion Chef™ instrument's IP address as shown below. (This isa blank file initially and is intended for custom firewall configuration.)

IMPORTANT! Only edit the /etc/iptables.custom file and not /etc/iptables.rules,since /etc/iptables.rules is overwritten when software is updated.

2. Enter "sudo nano /etc/iptables.custom" and add the three lines below:• *filter• -A INPUT -s xxx.xxx.xxx.xxx -j ACCEPT• COMMIT

Create anexception

Appendix C Ion Chef™ and Torrent Server network setupEditing the Torrent Server firewall tables to allow the LAN port IP C


Note: -A INPUT -s xxx.xxx.xxx.xxx -j ACCEPT adds the rule to theformat. Enter the IP address of the Ion Chef™ instrument in place of"xxx.xxx.xxx.xxx". When finished editing, save changes by pressing CTRL +O and CTRL + X.

3. Follow this command by manually applying the change:sudo iptables-restore --noflush < /etc/iptables.customNote: You can also list the firewall rules with: sudo iptables-L.

If this command returns with no output, it means it succeeded.

4. If the Ion Chef™ instrument is getting a DHCP address from your network,please create the exception for the entire DHCP subnet. The example belowassumes that the DHCP address for the Ion Chef™ instrument is 172.16.48.15 andthe DHCP subnet range is 172.16.48.1 to 172.16.48.254. Add these 3 lines:

• *filter• -A INPUT -s 172.16.48.0/24 -j ACCEPT• COMMIT

5. Restart networking by running this command: sudo /etc/init.d/networking restart.

6. If you want to repeat this process and completely drop all rules and start fresh,enter:

• sudo iptables-F• sudo iptables-restore</etc/iptables.rules• sudo ptables-restore--noflush</etc/iptables.custom

7. If a rule is ever removed from either of these files, this would remove it from theactive firewall rules because the iptables-restore command does notoverwrite rules, it appends them to all active rules.

Appendix C Ion Chef™ and Torrent Server network setupEditing the Torrent Server firewall tables to allow the LAN port IPC


Enable Planned Run sharing among Torrent Servers

New in Torrent Suite™ v4.4, Planned Runs created on one Torrent Server can now betransferred to another Torrent Server if the sequencer connected directly to the serveris offline or busy. Possible configurations include:

Ion Chef™ instrument 1 → Torrent Server 1 ↔ Torrent Server 2 → Sequencer 2

Ion Chef™ instrument 1 → Torrent Server 1 ↔ Torrent Server 3 → Sequencer 3

Network

TS 1(origin)

IC 2IC 1 Proton 1 Proton 2 IC 3

TS 2(destination)

TS 3(destination)

Proton 3

Requirements include:• All Torrent Servers must be running the same software version.• All Torrent Servers must have the same genomic reference, barcode set, BED files,

Variant Caller config files, etc. All Torrent Servers must be on the same subnet.

Appendix C Ion Chef™ and Torrent Server network setupEnable Planned Run sharing among Torrent Servers C


1. On the origin server (e.g., TS1) Site administration page, scroll down and selectShared servers. The Select shared server to change window appears.

2. If you are adding your destination server for the first time, click Add sharedserver.

3. Define your destination server.a. Enter the name, address (can be IP address), user name and password for

the destination server.

b. Click Active if you want this server enabled for sharing.

c. (Optional) Add a comment.

d. Click one of the Save options.

4. (Optional) If you want to configure the origin Torrent Server to also be adestination server, you must go to another server and repeat these steps to setthe origin server as a destination server. Once the Torrent Servers are configured,you or a user can now transfer Planned Runs between Torrent Servers.

Set up ServerNetwork (adminaction)

Appendix C Ion Chef™ and Torrent Server network setupEnable Planned Run sharing among Torrent ServersC


Transfer a Planned Run

You can execute a Planned Run on the Ion Chef™ instrument connected to the originserver, and then, when the run is complete, transfer the Planned Run to a destinationserver for sequencing.

Alternatively, you can create the Planned Run on an origin server and then transfer itto a destination server for both the Ion Chef™ run and the sequencing run.

1. Using the Torrent Browser on the origin Torrent Server, go to Plan4PlannedRun List.

2. Open the Gear menu of the Planned Run you want to transfer, and selectTransfer. Then select the destination Torrent Server.

3. A confirmation window appears. Check the information, then click Transfer.

Note: You can no longer access this Planned Run on the origin server after it hastransferred.

Appendix C Ion Chef™ and Torrent Server network setupTransfer a Planned Run C


4. A status window displays the results of the transfer:• The green box lists the samples successfully processed and the required

target BED files found on the destination server.• The red box lists any required BED files or plugins that are not present on

the destination server. To successfully perform the run, you will need to editthe transferred Planned Run on the destination server and manually add themissing BED files or plugins.

5. To edit the transferred Planned Run and add missing files:a. Download required files using the References tab of the Torrent browser of

the destination server

b. Go to the Edit Plan wizard of the transferred Planned Run by selecting Editon the gear pull-down menu to the right of the Planned Run.

c. Select the files or plugins as needed, then click Update Plan.

Note: You can also navigate to the Edit Plan wizard by clicking the Edit testplan link in the status page above.

Appendix C Ion Chef™ and Torrent Server network setupTransfer a Planned RunC


Undo a Planned Run transfer (administrator)

1. On the destination server, delete the transferred Planned Run from either thePlanned Run page or the admin page.

2. On the origin server, locate the plan on the /admin/rundb/plannedexperiment/page, uncheck PlanExecuted and change PlanStatus to Planned.

Undo a Planned Run transfer (user)

If you transferred a Planned Run in error, you can transfer it back to the origin serveror to another server.

1. On the destination Torrent Server, navigate to Plan4Planned Run List andlocate the transferred Planned Run.

2. From the Gear menu of the Planned Run, select Transfer, then select theTorrent Server to which you wish to transfer the run.

Appendix C Ion Chef™ and Torrent Server network setupUndo a Planned Run transfer (administrator) C


Safety

■ Symbols on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

■ Location of safety labels on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

■ Safety information for instruments not manufactured by ThermoFisher Scientific . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

■ Instrument safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

■ Safety and electromagnetic compatibility (EMC) standards . . . . . . . . . . . . . . . 137

■ Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

■ Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

WARNING! GENERAL SAFETY. Using this product in a manner not specifiedin the user documentation may result in personal injury or damage to theinstrument or device. Ensure that anyone using this product has receivedinstructions in general safety practices for laboratories and the safetyinformation provided in this document.

· Before using an instrument or device, read and understand the safetyinformation provided in the user documentation provided by themanufacturer of the instrument or device.

· Before handling chemicals, read and understand all applicable Safety DataSheets (SDSs) and use appropriate personal protective equipment (gloves,gowns, eye protection, etc). To obtain SDSs, see the “Documentation andSupport” section in this document.

D


Symbols on this instrument

Symbols may be found on the instrument to warn against potential hazards or conveyimportant safety information. In this document, the hazard symbol is used along withone of the following user attention words:

• CAUTION! – Indicates a potentially hazardous situation that, if not avoided,may result in minor or moderate injury. It may also be used to alert againstunsafe practices.

• WARNING! – Indicates a potentially hazardous situation that, if not avoided,could result in death or serious injury.

• DANGER! – Indicates an imminently hazardous situation that, if not avoided,will result in death or serious injury.

Symbol English Français

Caution, risk of danger

Consult the manual for further safetyinformation.

Attention, risque de danger

Consulter le manuel pour d’autresrenseignements de sécurité.

Caution, risk of electrical shock Attention, risque de choc électrique

Caution, hot surface Attention, surface chaude

Ultraviolet light Rayonnement ultraviolet

On/Off On/Off (marche/arrêt)

Protective conductor terminal (mainground)

Borne de conducteur de protection(mise à la terre principale)

Appendix D SafetySymbols on this instrument D


Symbol English Français

Do not dispose of this product inunsorted municipal waste

CAUTION! To minimizenegative environmentalimpact from disposal ofelectronic waste, do notdispose of electronic waste inunsorted municipal waste.Follow local municipal wasteordinances for properdisposal provision andcontact customer service forinformation aboutresponsible disposal options.

Ne pas éliminer ce produit avec lesdéchets usuels non soumis au trisélectif.

CAUTION! Pour minimiserles conséquences négativessur l’environnement à la suitede l’élimination de déchetsélectroniques, ne pas élimin-er ce déchet électroniqueavec les déchets usuels nonsoumis au tri sélectif. Se con-former aux ordonnances lo-cales sur les déchets munici-paux pour les dispositionsd’élimination et communi-quer avec le service à la cli-entèle pour des renseigne-ments sur les options d’élimi-nation responsable.

The following table applies only to the Ion Chef™ Instrument.

Conformitymark Description

Indicates conformity with safety requirements for Canada and U.S.A.

Indicates conformity with European Union requirements for safety andelectromagnetic compatibility.

Indicates conformity with Australian standards for electromagneticcompatibility.

Conformitysymbols

Appendix D SafetySymbols on this instrumentD


Location of safety labels on this instrument

Additional text may be used with one of the symbols described above when morespecific information is needed to avoid exposure to a hazard. See the following tablefor safety alerts found on the instrument.

English French translation Location onInstrument

CAUTION! Hazardouschemicals. Read the SafetyData Sheets (SDSs) beforehandling.

ATTENTION! Produits chimiquesdangereux. Lire les fichessignalétiques (FS) avant demanipuler les produits.

Back panel ofthe instrument.

CAUTION! Hazardouswaste. Refer to SDS(s) andlocal regulations forhandling and disposal.

ATTENTION! Déchets dangereux.Lire les fiches signalétiques (FS)et la réglementation localeassociées à la manipulation et àl’élimination des déchets.

Safety information for instruments not manufactured by ThermoFisher Scientific

Some of the accessories provided as part of the instrument system are not designed orbuilt by Thermo Fisher Scientific. Consult the manufacturer's documentation for theinformation needed for the safe use of these products.

Appendix D SafetyLocation of safety labels on this instrument D


Instrument safety

CAUTION! Do not remove instrument protective covers. If you remove theprotective instrument panels or disable interlock devices, you may be exposedto serious hazards including, but not limited to, severe electrical shock, laserexposure, crushing, or chemical exposure.

CAUTION! Moving and Lifting Injury. The instrument is to be moved andpositioned only by the personnel or vendor specified in the applicable sitepreparation guide. Improper lifting can cause painful and permanent backinjury.

Things to consider before lifting or moving the instrument or accessories:· Depending on the weight, moving or lifting may require two or more

persons.· If you decide to lift or move the instrument after it has been installed, do not

attempt to do so without the assistance of others, the use of appropriatemoving equipment, and proper lifting techniques.

· Ensure you have a secure, comfortable grip on the instrument or accessory.· Make sure that the path from where the object is to where it is being moved

is clear of obstructions.· Do not lift an object and twist your torso at the same time. Keep your spine

in a good neutral position while lifting with your legs.· Participants should coordinate lift and move intentions with each other

before lifting and carrying.· For smaller packages, rather than lifting the object from the packing box,

carefully tilt the box on its side and hold it stationary while someone elseslides the contents out of the box.

CAUTION! Moving Parts. Moving parts can crush, pinch and cut. Keep handsclear of moving parts while operating the instrument. Disconnect power beforeservicing.

General

Physical injury

Appendix D SafetyInstrument safetyD


WARNING! Ensure appropriate electrical supply. For safe operation of theinstrument:

· Plug the system into a properly grounded receptacle with adequate currentcapacity.

· Ensure the electrical supply is of suitable voltage.· Never operate the instrument with the ground disconnected. Grounding

continuity is required for safe operation of the instrument.

WARNING! Power Supply Line Cords. Use properly configured and approvedline cords for the power supply in your facility.

WARNING! Disconnecting Power. To fully disconnect power either detach orunplug the power cord, positioning the instrument such that the power cord isaccessible.

CAUTION! Cleaning and Decontamination. Use only the cleaning anddecontamination methods specified in the manufacturer's user documentation.It is the responsibility of the operator (or other responsible person) to ensurethe following requirements are met:

· No decontamination or cleaning agents are used that could cause aHAZARD as a result of a reaction with parts of the equipment or withmaterial contained in the equipment.

· The instrument is properly decontaminated a) if hazardous material isspilled onto or into the equipment, and/or b) prior to having the instrumentserviced at your facility or sending the instrument for repair, maintenance,trade-in, disposal, or termination of a loan (decontamination forms may berequested from customer service).

· Before using any cleaning or decontamination methods (except thoserecommended by the manufacturer), users should confirm with themanufacturer that the proposed method will not damage the equipment.

The Ion Chef™ System uses a UV lamp which emits light at 254 nm. Under normaloperating conditions, the UV lamp is powered on when performing the cleaningprotocol. Safety interlocks are used to ensure that the UV lamp is not powered whenthe door is open.

Safety and electromagnetic compatibility (EMC) standards

The instrument design and manufacture complies with the standards andrequirements for safety and electromagnetic compatibility as noted in the followingtable:

Electrical

Cleaning anddecontamination

Ultraviolet (UV)Safety

Appendix D SafetySafety and electromagnetic compatibility (EMC) standards D


Reference Description

EU Directive 2006/95/EC European Union “Low Voltage Directive”

IEC 61010-1

EN 61010-1

UL 61010-1

CSA C22.2 No. 61010-1

Safety requirements for electrical equipment formeasurement, control, and laboratory use – Part 1: Generalrequirements

IEC 61010-2-010

EN 61010-2-010

Safety requirements for electrical equipment formeasurement, control and laboratory use – Part 2-010:Particular requirements for laboratory equipment for theheating of materials

IEC 61010-2-020

EN 61010-2-020

Safety requirements for electrical equipment formeasurement, control and laboratory use – Part 2-020:Particular requirements for laboratory centrifuges

IEC 61010-2-081

EN 61010-2-081

Safety requirements for electrical equipment formeasurement, control and laboratory use – Part 2-081:Particular requirements for automatic and semi-automaticlaboratory equipment for analysis and other purposes


Directive 2004/108/EC European Union “EMC Directive”

FCC Part 15 U.S. Standard “Industrial, Scientific, and Medical Equipment”

AS/NZS 2064 Limits and Methods of Measurement of ElectromagneticDisturbance Characteristics of Industrial, Scientific, andMedical (ISM) Radiofrequency Equipment

ICES-001, Issue 3 Industrial, Scientific and Medical (ISM) Radio FrequencyGenerators


Directive 2012/19/EU European Union “WEEE Directive” – Waste electrical andelectronic equipment

Safety

EMC

Environmentaldesign

Appendix D SafetySafety and electromagnetic compatibility (EMC) standardsD


Chemical safety

WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,ensure laboratory personnel read and practice the general safety guidelines forchemical usage, storage, and waste provided below, and consult the relevantSDS for specific precautions and instructions:· Read and understand the Safety Data Sheets (SDSs) provided by the

chemical manufacturer before you store, handle, or work with any chemicalsor hazardous materials. To obtain SDSs, see the “Documentation andSupport” section in this document.

· Minimize contact with chemicals. Wear appropriate personal protectiveequipment when handling chemicals (for example, safety glasses, gloves, orprotective clothing).

· Minimize the inhalation of chemicals. Do not leave chemical containers open.Use only with adequate ventilation (for example, fume hood).

· Check regularly for chemical leaks or spills. If a leak or spill occurs, followthe manufacturer's cleanup procedures as recommended in the SDS.

· Handle chemical wastes in a fume hood.· Ensure use of primary and secondary waste containers. (A primary waste

container holds the immediate waste. A secondary container contains spillsor leaks from the primary container. Both containers must be compatiblewith the waste material and meet federal, state, and local requirements forcontainer storage.)

· After emptying a waste container, seal it with the cap provided.· Characterize (by analysis if necessary) the waste generated by the particular

applications, reagents, and substrates used in your laboratory.· Ensure that the waste is stored, transferred, transported, and disposed of

according to all local, state/provincial, and/or national regulations.· IMPORTANT! Radioactive or biohazardous materials may require special

handling, and disposal limitations may apply.

WARNING! HAZARDOUS WASTE (from instruments). Waste produced bythe instrument is potentially hazardous. Follow the guidelines noted in thepreceding General Chemical Handling warning.

Appendix D SafetyChemical safety D


Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,infectious agents, and blood of humans and other animals have the potential totransmit infectious diseases. All work should be conducted in properlyequipped facilities using the appropriate safety equipment (for example,physical containment devices). Safety equipment also may include items forpersonal protection, such as gloves, coats, gowns, shoe covers, boots,respirators, face shields, safety glasses, or goggles. Individuals should betrained according to applicable regulatory and company/ institutionrequirements before working with potentially biohazardous materials. Followall applicable local, state/provincial, and/or national regulations. The followingreferences provide general guidelines when handling biological samples inlaboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiologicaland Biomedical Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC)21-1112, Revised December 2009; found at:www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

· World Health Organization, Laboratory Biosafety Manual, 3rd Edition,WHO/CDS/CSR/LYO/2004.11; found at:www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Appendix D SafetyBiological hazard safetyD


http://www.cdc.gov/biosafety/publications/bmbl5/BMBL.pdf

http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf

Documentation and support

Customer and technical support

Visit www.lifetechnologies.com/support for the latest in services and support,including:

• Worldwide contact telephone numbers• Product support, including:

– Product FAQs– Software, patches, and updates

• Order and web support• Product documentation, including:

– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale found on LifeTechnologies' website at www.lifetechnologies.com/termsandconditions. If you haveany questions, please contact Life Technologies at www.lifetechnologies.com/support.


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Ion PI IC 200 Kit - Thermo Fisher Scientifictools.thermofisher.com/content/sfs/manuals/MAN0010078_Ion_PI_IC_200_UG.pdfThe Ion PI™ IC 200 Kit (Cat. no. 4488377) contains all materials

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