Ion AmpliSeq Library Preparation on the Ion Chef System ... · PDF fileIon AmpliSeq™ Library Preparation on the Ion Chef ... Chef‑ready Kit (Cat. No. A32841) 2 132/133 19/22 4

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

Ion AmpliSeq™ Library Preparation on the IonChef™ SystemUSER GUIDE

for use with:Ion AmpliSeq™ Kit for Chef DL8Ion AmpliSeq™ Chef-ready KitsOncomine™ Research Assay Chef-ready KitsIon AmpliSeq™ Direct FFPE DNA KitIon AmpliSeq™ On‑Demand PanelsIon AmpliSeq™ Ready-to-Use PanelsIon AmpliSeq™ Made-to-Order and Community PanelsIon AmpliSeq™ Sample ID PanelIon Chef™ System

Catalog Numbers A29024, A35121, A32914, A29998, A31446, A32841, A31133, A31136, 4479790,4484177Publication Number MAN0013432

Revision E.0

Manufacturer: Multiple Life Technologies Corporation manufacturing sites are responsible for manufacturing the products associated withthe workflow covered in this guide.

Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288

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 YOURUSE OF IT.

Revision history: Pub. No. MAN0013432

Revision Date DescriptionE.0 16 May 2017 • Support added for Ion AmpliSeq™ On‑Demand Panels

• Last two rows of the amplification cycle table merged, anneal/extension timecolumn added to table, and anneal/extension time recommendations modified forhigher plexy panels (see “Start the Ion Chef™ run“ on page 28)

D.0 14 March 2017 • Chapter structure reorganized for ease of use

• Reverse transcription reaction conditions for use of the SuperScript™ IV VILO™

Master Mix added

• List of Chef-ready kits updated with the Ion AmpliSeq™ Transcriptome HumanGene Expression Panel, Oncomine™ BRCA Research Assay, and Oncomine™

Immune Response Research Assay Chef-ready Kits

• Guidance added for using the Ion AmpliSeq™ Comprehensive Cancer Panel as a2‑primer pool panel with the Ion AmpliSeq™ Kit for Chef DL8 (see “Tips“ onpage 41)

• Ion Chip capacity table for Ion AmpliSeq™ DNA libraries updated

C.0 6 July 2016 • List of Chef-ready panel kits updated with the Ion AmpliSeq™ Cancer HotspotResearch Panel v2: Chef-ready Kit

• Added support for the Ion AmpliSeq™ Direct FFPE DNA Kit

B.0 2 November 2015 • Updated guidance for runs with 5 or fewer samples

• Updated guidance for removing library tube from instrument after a run

A.0 31 August 2015 New user guide, with detailed instructions for using the Ion AmpliSeq™ Kit for Chef DL8with the Ion Chef™ Instrument to prepare Ion AmpliSeq™ libraries for templating andsequencing on the Ion PGM™, Ion Proton™, or Ion S5™ System

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you acceptthe terms and conditions of all applicable Limited Use Label Licenses.Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registeredtrademark of Roche Molecular Systems, Inc., used under permission and license.

©2017 Thermo Fisher Scientific Inc. All rights reserved.

Contents

■ CHAPTER 1 Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Ion AmpliSeq™ Kit for Chef DL8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Ion AmpliSeq™ and Oncomine™ Research Assay Chef-ready Kits . . . . . . . . . . . . . . . . . . . . . . 9

Ion AmpliSeq™ Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Ion AmpliSeq™ On‑Demand Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Ion AmpliSeq™ Ready-to-Use Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Ion AmpliSeq™ Made-to-Order and Community Panels . . . . . . . . . . . . . . . . . . . . . . . . . 11Ion AmpliSeq™ Sample ID Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Ion AmpliSeq™ Direct FFPE DNA Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Recommended materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■ CHAPTER 2 Prepare DNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Isolate and dilute DNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Guidelines for isolating and quantifying gDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Prepare DNA with the Ion AmpliSeq™ Direct FFPE DNA Kit . . . . . . . . . . . . . . . . . . . . . . 15Dilute the DNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Add DNA to the IonCode™ PCR plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

■ CHAPTER 3 Prepare RNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Isolate and dilute RNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Guidelines for isolating and quantifying RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Dilute the RNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Reverse transcribe RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 3

■ CHAPTER 4 Run the Ion Chef™ System .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Thaw the reagents and prepare the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

(Optional) Create a Sample Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Set up the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Add Ion AmpliSeq™ 2X Primer Pools to Positions A and B of theReagents cartridge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25Load the Ion Chef™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Start the Ion Chef™ run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Unload the Ion Chef™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33(Optional) Combine libraries from multiple Ion Chef™ runs . . . . . . . . . . . . . . . . . . . . . . 34

Clean the Ion Chef™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35About the cleaning protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Clean the Ion Chef™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Proceed to an Ion Chef™ template run and create a Planned Run template . . . . . . . . . . . . 37

■ APPENDIX A Tips and troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Ion AmpliSeq™ Direct FFPE DNA Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Ion AmpliSeq™ library troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Ion Chef™ System setup and operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

■ APPENDIX B Supplemental procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Reverse transcribe RNA using the SuperScript™ VILO™ cDNA Synthesis Kit . . . . . . . . . . . . 47

(Optional) Remove deaminated bases from Direct FFPE DNA . . . . . . . . . . . . . . . . . . . . . . . . 49

(Optional) Qubit™ Fluorometer: Quantify the FFPE DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

■ APPENDIX C Strategies for combining Ion AmpliSeq™ libraries . . . 50

Combine barcoded libraries from multiple runs for sequencing on a single Ion chip . . . . 50

Combine libraries prepared using different panels for equal coverage . . . . . . . . . . . . . . . . 51

Combine unequal volumes of libraries for varying depth of coverage . . . . . . . . . . . . . . . . . 52

Ion Chip capacities for Ion AmpliSeq™ DNA libraries sequenced at equal depths . . . . . . . 53

Ion Chip capacities for Ion AmpliSeq™ RNA libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

■ APPENDIX D Ion AmpliSeq™ Made-to-Order Panels . . . . . . . . . . . . . . . . 55

Prepare primer pools from plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

■ APPENDIX E Ion AmpliSeq™ Sample ID Panel . . . . . . . . . . . . . . . . . . . . . . . 57

Using the Sample ID Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Contents

4 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide

■ APPENDIX F Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Torrent Coverage Analysis Plugin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Configure and run the Torrent Coverage Analysis Plugin . . . . . . . . . . . . . . . . . . . . . . . . 59Coverage Analysis Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

ampliSeqRNA Plugin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65Configure and run the ampliSeqRNA Plugin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

■ APPENDIX G Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Symbols on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67Conformity symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Location of safety labels on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

Safety information for instruments not manufactured by Thermo Fisher Scientific . . . . . 69

Instrument safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69Physical injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70Electrical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70Cleaning and decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71Ultraviolet (UV) Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Safety and electromagnetic compatibility (EMC) standards . . . . . . . . . . . . . . . . . . . . . . . . . . 71Safety compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71EMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72Environmental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Contents


Product information

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.

Product description

The Ion AmpliSeq™ Kit for Chef DL8 (DNA to Library, 8 samples/run) includes thereagents and materials for automated preparation of up to 8 barcoded Ion AmpliSeq™

libraries per Ion Chef™ run using a single 1- or 2-pool Ion AmpliSeq™ panel. The kitprovides reagents and materials sufficient for 4 runs, giving the user the ability toprepare up to 32 barcoded libraries. Fewer than 8 samples can be processed in a run,but a run consumes kit reagents for 8 samples regardless of sample number.

The Ion Chef™ Instrument sets up a target amplification reaction for each sample witheach primer pool, using a different barcode for each sample. Library concentration isnormalized to approximately 100 pM using Ion Library Equalizer™ technology, andlibraries, combined in a single tube, are ready to use in Ion Chef™ or Ion OneTouch™ 2template preparation reactions.

Use of the kit with the Ion Chef™ System leverages the automated fluid handling andPCR amplification of the system to prepare barcoded, purified, and equalized IonAmpliSeq™ libraries with less than 15 minutes of hands-on time. Total instrument runtime is approximately 7 hours.

The Ion AmpliSeq™ Kit for Chef DL8 is compatible with Torrent Suite™ Software 5.0and later. We recommend updating your Torrent Server, Ion Chef™, and Ion sequencerinstrument software to the latest available versions before using this kit.

1

Softwarecompatibility


Ion AmpliSeq™ Kit for Chef DL8

IMPORTANT! Store all consumables and cartridges under the recommendedconditions and in an upright position. Ion AmpliSeq™ Chef Solutions DL8 cartridgesare shipped at ambient temperature, but need to be stored at 2°C to 8°C upon arrival.

The Ion AmpliSeq™ Kit for Chef DL8 (Cat. No. A29024) contains materials requiredfor performing 4 Ion Chef™ runs, with up to 8 Ion AmpliSeq™ libraries prepared perrun. Upon arrival, inspect all consumables and contact Technical Support if any of thecomponents have been damaged during shipping.

Component Part No. Quantity perkit Storage

Ion AmpliSeq™ Chef Reagents DL8 A29025 4 cartridges –30°C to–10°C

Ion AmpliSeq™ Chef Solutions DL8 A29026 4 cartridges 2°C to 8°C[1]

Ion AmpliSeq™ Chef Supplies DL8 (per insert)

• Ion AmpliSeq™ Tip Cartridge L8

• PCR Frame Seal

• Enrichment Cartridge

A29027 1 box with4 inserts

15°C to 30°C

IonCode™ 0101–0132 in 96 Well PCR Plates(dried)

Set includes 4 PCR plates:

• IonCode™ 0101–0108 in 96 Well PCR Plate(red)

• IonCode™ 0109–0116 in 96 Well PCR Plate(yellow)

• IonCode™ 0117–0124 in 96 Well PCR Plate(green)

• IonCode™ 0125–0132 in 96 Well PCR Plate(blue)

A29028 1 set of4 plates

15°C to 30°C

[1] Ion AmpliSeq™ Chef Solutions DL8 cartridges are shipped at ambient temperature, but need to be stored at 2°C to 8°C upon arrival.

Chapter 1 Product informationIon AmpliSeq™ Kit for Chef DL8 1


1

2

3

4

5

6

8

7

9

Ion AmpliSeq™ Kit for Chef DL8 consumables1 Ion AmpliSeq™ Chef Reagents DL8 cartridge2 Ion AmpliSeq™ Chef Solutions DL8 cartridge3 Ion AmpliSeq™ Tip Cartridge L84 Enrichment Cartridge5 PCR Frame Seal

6 IonCode™ 0101–0108 in 96 Well PCR Plate (red)7 IonCode™ 0109–0116 in 96 Well PCR Plate (yellow)8 IonCode™ 0117–0124 in 96 Well PCR Plate (green)9 IonCode™ 0125–0132 in 96 Well PCR Plate (blue)

IMPORTANT! Use scissors to open the plastic bag containing the PCR Frame Seal.Tearing the bag open may damage the seal.

Chapter 1 Product informationIon AmpliSeq™ Kit for Chef DL81


Ion AmpliSeq™ and Oncomine™ Research Assay Chef-ready Kits

Ion AmpliSeq™ and Oncomine™ Research Assay Chef-ready Kits provide primerpanels and all of the reagents and supplies in a Ion AmpliSeq™ Kit for Chef DL8 toprepare 32 libraries on the Ion Chef™ System. The primer panels are supplied at theappropriate volume and concentration ready to load into an Ion AmpliSeq™ ChefReagents DL8 cartridge.

Kit Primerpools

Number ofprimer

pairs/pool

Amplification cycles forStandard/FFPE

Anneal & Extensiontime

Quantity Storage

DNA library Chef-ready Kits

Ion AmpliSeq™ Cancer HotspotResearch Panel v2, Chef-ready Kit(Cat. No. A32914)

1 207 18/21

4 minutes

32 libraries(4 runs)8 tubes

–30°C to–10°C

Ion AmpliSeq™ PharmacogenomicsResearch Panel, Chef-ready Kit(Cat. No. A29998)

1 119 21/24[1]

4 minutes

Oncomine™ BRCA Research AssayChef‑ready Kit (Cat. No. A32841)

2 132/133 19/22

4 minutes

RNA library Chef-ready Kits

Ion AmpliSeq™ Transcriptome HumanGene Expression Panel, Chef-ready Kit(Cat. No. A31446)

1 20,800 12/17[2]

16 minutes

32 libraries(4 runs)8 tubes

–30°C to–10°C

Oncomine™ Immune ResponseResearch Assay Chef‑ready Kit(Cat. No. A32928)

1 394 20/20

4 minutes

[1] We recommend using 2 additional cycles over the cycle recommendation on page 28 with this panel.[2] We recommend using 2 additional cycles over the cycle recommendation on page 28 for FFPE RNA with this panel.

Chapter 1 Product informationIon AmpliSeq™ and Oncomine™ Research Assay Chef-ready Kits 1


Ion AmpliSeq™ Panels

Ion AmpliSeq™ On-Demand Panels (Cat. No. A35121) are for use in inherited disease(germline mutation) research applications. Design and order an Ion AmpliSeq™

On-Demand Panel at AmpliSeq.com by selecting genes using a content selectionengine, or upload your own gene list. Each panel consists of two DNA primer pools.The number of primer pairs per pool depends on the genes you select. Ion AmpliSeq™

On-Demand Panels are for Research Use Only. Not for use in diagnostic procedures.

Panels are formatted for:• Manual library preparation using the Ion AmpliSeq™ Library Kit Plus (Cat. No.

4488990), or• Library preparation on the Ion Chef™ System using the Ion AmpliSeq™ Kit for

Chef DL8 (Cat. No. A29024)

Ion AmpliSeq™ On‑Demand Panelformat Cat. No.

Number ofreactions per

panel

Number ofgenes per panel

Manual library preparation[1]A33093 24 1−50

A33094 24 51−300

Automated library preparation onthe Ion Chef™ System

A33095 8 1−50

A33096 8 51−300

[1] See the Ion AmpliSeq™ Library Kit Plus User Guide (Pub. No. MAN0017003) for further information.

Visit www.ampliseq.com for a complete listing of Ion AmpliSeq™ Ready-to-UsePanels, ordered separately, compatible with the Ion AmpliSeq™ Kit for Chef DL8. Upto 8 libraries can be prepared in each run with a single panel composed of 1 or 2primer pools.

Ion AmpliSeq™

On‑DemandPanels

Ion AmpliSeq™

Ready-to-UsePanels

Chapter 1 Product informationIon AmpliSeq™ Panels1


https://www.ampliseq.com


Visit AmpliSeq.com to design and order Ion AmpliSeq™ Made-to-Order Panels, andto order Ion AmpliSeq™ Community Panels. Each order includes one or more pre-made pools of primer pairs at 2X or 5X concentration for use with standard IonAmpliSeq™ Library Kits or the Ion AmpliSeq™ Kit for Chef DL8.

Average primerpairs per pool in aMade-to-Order orCommunity Panel

Panels with 1 primerpool[1]

Panels with 2 primerpools Storage

Ion AmpliSeq™ Made-to-Order and Community DNA Panels (2X primer pools)

£96 primer pairs 5 tubes, ~1500 µL each(25 runs, 200 libraries)

10 tubes, ~1500 µL each(50 runs, 400 libraries)

–30°C to–10°C

>96 primer pairs 20 tubes, ~1500 µL each(100 runs, 800 libraries)

40 tubes, ~1500 µL each(200 runs, 1600

libraries)

Ion AmpliSeq™ Made-to-Order and Community RNA Panels (5X primer pools)

12–1,200 3 tubes, ~1500 µL each(~37 runs, 300 libraries)

6 tubes, ~1500 µL each(~75 runs, 600 libraries)

–30°C to–10°C

[1] Single pool panels require twice as much volume and therefore generate half the number of libraries per order.

The Ion AmpliSeq™ Sample ID Panel can be used in conjunction with all IonAmpliSeq™ Chef-ready, Ready-to-Use, Made-to-Order, and Community Panelstargeting human genomic DNA. (Add 15 µL of the Ion AmpliSeq™ Sample ID Paneland 135 µL of the primer panel to one or both Primer Panel Tubes.) The IonAmpliSeq™ Sample ID Panel (Cat. No. 4479790) is ordered separately.

Component Species Conc. QuantityNo. of

primerpairs

Storage

Ion AmpliSeq™ SampleID Panel

human(gDNA)

20X 1 tube 9 –30°C to–10°C

Ion AmpliSeq™ Direct FFPE DNA Kit

The Ion AmpliSeq™ Direct FFPE DNA Kit provides an optional method for directanalysis of DNA from 8 (Cat. No. A31133) or 96 (Cat. No. A31136) unstained, slide-mounted, formalin-fixed, paraffin-embedded (FFPE) tissue samples for downstreamlibrary preparation without nucleic acid isolation or quantification.

Component Cat. No. A31133(8 rxns)

Cat. No. A31136(96 rxns)

Storage

Transfer Solution (purple cap) 240 µL 3 × 960 µL 2°C to 8°C

Direct Reagent (orange cap) 170 µL 3 × 675 µL

Ion AmpliSeq™

Made-to-Orderand CommunityPanels

Ion AmpliSeq™

Sample ID Panel

Chapter 1 Product informationIon AmpliSeq™ Direct FFPE DNA Kit 1



Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com.MLS: Fisher Scientific (fisherscientific.com) or other major laboratory supplier.

Item Source

Ion Chef™ System 4884177

P20 and P200 µL pipettors, and filtered tips fisherscientific.com

(Optional) Non-interruptible Power Supply (UPS)[1] MLS

Nuclease-free Water AM9932

Low TE (10 mM Tris, 0.1 mM EDTA, pH 8.0) 50-843-203(fisherscientific.com)

Required for libraries from RNA

SuperScript™ IV VILO™ Master Mix or

SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme or

SuperScript™ VILO™ cDNA Synthesis Kit (recommended for IonAmpliSeq™ Transcriptome Human Gene Expression Panel)

11756050

11766050

11754050

MicroAmp™ Clear Adhesive Film 4306311

MicroAmp™ Optical Film Compression Pad 4312639

Veriti™ Thermal Cycler or equivalent 4375786

[1] For laboratories that experience frequent power outages or line voltage fluctuations, we recommend that you use an non-interruptible power supply that provides 2500 W output or higher.

Recommended materials and equipment

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

Item Source

Additional equipment

Fisher Scientific™ Mini Plate Spinner Centrifuge, or equivalent96-well plate centrifuge

14‑100‑143(fisherscientific.com)

Recommended for nucleic acid isolation

Ion AmpliSeq™ Direct FFPE DNA Kit A31133

RecoverAll™ Total Nucleic Acid Isolation Kit AM1975

MagMAX™ FFPE Total Nucleic Acid Isolation Kit 4463365

PureLink™ Genomic DNA Mini Kit K182001

PureLink™ RNA Mini Kit 12183018A

Chapter 1 Product informationRequired materials not supplied1


http://www.thermofisher.com

http://fisherscientific.com



http://www.thermofisher.com


Item Source

Recommended for nucleic acid quantification

TaqMan® RNase P Detection Reagents Kit (Recommended forgDNA)

4316831

Qubit™ 3.0 Fluorometer

Qubit™ dsDNA HS Assay Kit (DNA), or

Qubit™ RNA HS Assay Kit (RNA)

Q33216

Q32851, Q32854

Q32852, Q32855

(Optional) Ion Library TaqMan® Quantitation Kit[1] 4468802

Materials recommended for the Ion AmpliSeq™ Direct FFPE DNA Kit

One of the following:

• SimpliAmp™ Thermal Cycler

• AB™ 2720 Thermal Cycler

• Veriti™ 96-well Thermal Cycler

• ProFlex™ 96-Well PCR System

• GeneAmp™ PCR System 9700[2] or Dual 96-well ThermalCycler

See web productpages

MicroAmp™ Optical 96-well Reaction Plate N8010560

4306737(with barcode)

MicroAmp™ Clear Adhesive Film 4306311

MicroAmp™ Optical Film Compression Pad 4312639

(Optional) Uracil DNA Glycosylase (UDG) 18054015, EN0361

[1] Recommended for checking concentration of combined library.[2] Supported but no longer available for purchase.

Chapter 1 Product informationRecommended materials and equipment 1


Workflow

The following workflow summarizes the preparation of DNA or RNA Ion AmpliSeq™

libraries using the Ion AmpliSeq™ Kit for Chef DL8 and the Ion Chef™ System.

Isolate and dilute DNA samples

q

Isolate and dilute RNA samples

q

Add DNA to the IonCode™ PCR plate Reverse transcribe RNA using theSuperScript™ VILO™ cDNA Synthesis Kit

q

(Optional) Create a Sample Set

q

Add Ion AmpliSeq™ 2X Primer Pools to Positions A and B of the Reagents cartridge

q

Load the Ion Chef™ Instrument

q

Start the Ion Chef™ run

q

Unload the Ion Chef™ Instrument

Chapter 1 Product informationWorkflow1


Prepare DNA samples

■ Isolate and dilute DNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■ Add DNA to the IonCode™ PCR plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

For information on preparing RNA samples, go to Chapter 3, “Prepare RNAsamples“.

Isolate and dilute DNA samples

• High-quality gDNA can be prepared and quantified using commerciallyavailable kits. See “Required materials not supplied“ on page 12 for kits that arerecommended for isolating gDNA from fresh and FFPE (Formalin-fixed, paraffin-embedded) samples.

• We recommend the TaqMan® RNase P Detection Reagents Kit (Cat. No. 4316831)for quantifying amplifiable human genomic DNA. See Demonstrated Protocol:Sample Quantification for Ion AmpliSeq™ Library Preparation Using the TaqMan®

RNAse P Detection Reagents Kit (Pub. No. MAN0007732), available at thermofisher.com.

• The Qubit™ dsDNA HS Assay Kit (Cat. No. Q32851 or Q32854) can also be usedfor quantification, particularly for FFPE DNA, and highly degraded DNAsamples.

• Methods such as densitometry (for example, NanoDrop™ spectrophotometers)are not recommended, because they are not specific for gDNA, and are thereforesensitive to small fragments of hydrolyzed RNA. Using densitometry can lead tooverestimation of the sample gDNA concentration, under-seeding of the targetamplification reaction, low library yields, and poor chip loading.

• The Direct FFPE DNA preparation can be stored for up to 6 months at -20°Cbefore library preparation.

The Ion AmpliSeq™ Direct FFPE DNA Kit (Cat. Nos. A31133 and A31136) bypassesnucleic acid isolation when preparing libraries from FFPE sections on slides. Use thefollowing protocol with the Ion AmpliSeq™ Direct FFPE DNA Kit to prepare DNAfrom FFPE tissue sections suitable for use in Ion AmpliSeq™ library preparation.

Prepare reagents

• Equilibrate Transfer Solution to room temperature (15–30°C) before use.

• Keep Direct Reagent on ice prior to use.

2

Guidelines forisolating andquantifying gDNA

Prepare DNA withthe Ion AmpliSeq™

Direct FFPE DNAKit


https://www.thermofisher.com

Prepare FFPE DNA

The recommended tissue area to be usedfor this protocol is 4−100 mm2 from a5−10 µm thick unstained section mountedon a slide. The shaded squares on theslide at right represent a 4 mm2 area outof a total gridded area of 100 mm2.Deparaffinization is not required. Ifdesired, scrape unwanted tissue from theslide before transfer.

1. For each sample, pipet 30 µL of Transfer Solution into a single well of a 96-wellPCR plate.

2. Using a single 20-µL pipette tip for each sample:a. Pipet 2–10 µL of the Transfer Solution from the well onto the region of

interest of the FFPE tissue section mounted on a slide.

b. Using the same 20-µL pipette tip, spread the Transfer Solution to ensurecomplete coverage of the region of interest, then scrape and break up thetissue with the pipette tip. The tissue should be a slurry of fine particles inthe Transfer Solution.

3. Pipet the slurry from the slide back into the same well of the 96-well platecontaining Transfer Solution.

4. Pipette the slurry up and down at least 5 times, leaving as much tissue aspossible in the 96-well plate.

5. If needed, use the same tip to repeat steps 2–4, transferring as much of the regionof interest as possible into the 96-well plate.

Note: Although the final volume of Transfer Solution remaining in the 96-wellplate can vary, no volumetric adjustment is required.

6. Add 21 µL of Direct Reagent to each well containing sample in the 96-well plate.

7. Set a pipette to 30 µL, then mix the Direct Reagent and slurry by pipetting upand down 10 times.

8. Seal the plate with a MicroAmp™ Adhesive Film, then verify that the contents areat the bottom of each well of the 96-well plate.

Note: If needed, gently tap the plate on a hard flat surface to collect the contentsat the bottom of the wells.

9. Place a compression pad on the plate, load the plate into the thermal cycler, thenrun the following program:

Temperature Time

65°C 15 minutes

20°C Hold (for up to 30 minutes)

Proceed to “Remove an aliquot for library preparation“.

0 5 10mm

0

5

10

mm

Standard microscope slide, 25 × 75 mm.

Chapter 2 Prepare DNA samplesIsolate and dilute DNA samples2


Note: See “(Optional) Remove deaminated bases from Direct FFPE DNA“ onpage 49 for an optional Uracil DNA Glycosylase (UDG) treatment procedure.Sample DNA concentration can be evaluated using an optional Qubit™ fluorometryprotocol. See “(Optional) Qubit™ Fluorometer: Quantify the FFPE DNA“ on page 49.

Remove an aliquot for library preparation

1. Set a 20-µL pipette to 15 µL, depressthe plunger to the first stop andinsert the pipet tip into the lower(aqueous) phase, then pipet up anddown to mix the sample.

Note: Mixing the sample beforeremoval ensures a homogeneoussample before removing aliquots.Avoid disturbing the upper phase and interface while mixing.

2. Remove 15 µL of the lower phase,then transfer the sample to theappropriate column 1 well of anIonCode™ 96-well PCR plate.

Note: Avoid pipetting the upperphase that contains the TransferSolution.

3. Carefully inspect each transferred sample aliquot for air bubbles. Remove any airbubbles by gently pipetting up and down.

Proceed to “Thaw the reagents and prepare the instrument“ on page 22.

Note:· You can thaw reagents, prepare the instrument, and add primer pools to the

Reagents cartridge ahead of time so that samples are ready to be loaded into theinstrument immediately after transfer to the IonCode™ 96-well PCR plate.

· The Direct FFPE DNA preparation can be stored for up to 6 months at −20°C beforelibrary preparation.

Dilute samples to 0.67 ng/µL with Nuclease-free Water. You need to prepare 15 µL ofeach diluted sample (10 ng) to make an Ion AmpliSeq™ Chef library.

Note:· If you choose to use more gDNA, increase the concentration, not the volume. For

example, to apply 100 ng of gDNA per library, use 15 µL of gDNA at 6.7 ng/µL.· If you are using Direct FFPE DNA, do not dilute the samples.

Dilute the DNAsamples

Chapter 2 Prepare DNA samplesIsolate and dilute DNA samples 2


Add DNA to the IonCode™ PCR plate

1. Remove and discard the plate seal from an IonCode™ 96 Well PCR Plate.

2. Pipet 15 µL of each gDNA sample (0.67 ng/µL, 10 ng), or Direct FFPE DNAsample, into wells A1 to H1 of the plate.

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

B

C

D

E

F

G

H

21

1 Each column 1 well contains 15 µL of diluted gDNA sample (0.67 ng/µL, 10 ng total),Direct FFPE DNA, or Nuclease-free Water as non-template control.

2 Each column 6 well contains a dried-down IonCode™ barcode. The lowest barcodenumber is in A6, and the highest barcode number is in H6. All appear light blue in theactual plates.

3. Carefully inspect each well for air bubbles. Remove any air bubbles by gentlepipetting. Alternatively, centrifuge the plate briefly in a plate centrifuge.

4. If you are processing fewer than 8 samples, it is preferable to add replicates orpositive control samples to the run. Otherwise, pipet 15 µL of Nuclease-freeWater as non-template control into column 1 wells that do not contain a DNAsample.

Note: If processing 5 or fewer samples, we recommend that you quantify theoutput combined library by qPCR to ensure that an optimal concentration is usedin templating reactions.

IMPORTANT! If using the Ion AmpliSeq™ Direct FFPE DNA Kit, start theIon Chef™ run within 10 minutes of transferring the last sample slurry to theIonCode™ 96-well PCR plate. If ≥10 minutes has elapsed, pipet each sampleslurry up and down at least 5 times to mix, load the IonCode™ 96-well PCR plateonto the Ion Chef™ Instrument, then start the run. We recommend that you thawreagents, prepare the instrument, and add primer pools to the Reagents cartridgeahead of time so that samples are ready to be loaded into the instrumentimmediately after transfer to the IonCode™ 96-well PCR plate.


Chapter 2 Prepare DNA samplesAdd DNA to the IonCode™ PCR plate2


Prepare RNA samples

■ Isolate and dilute RNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

■ Reverse transcribe RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

For information on preparing DNA samples, go to Chapter 2, “Prepare DNAsamples“.

Isolate and dilute RNA samples

• High-quality RNA can be prepared and quantified using commercially availablekits. See “Required materials not supplied“ on page 12 for kits recommended forisolating RNA from fresh and FFPE samples.

• We recommend the Qubit™ RNA HS Assay Kit (Cat. No. Q32855) for quantifyingRNA.

• Methods such as densitometry (for example, NanoDrop™ spectrophotometers)are not recommended, because they are sensitive to small fragments ofhydrolyzed RNA. Using densitometry can lead to overestimation of the sampleRNA concentration, under-seeding of the target amplification reaction, lowlibrary yields, and poor chip loading.

Dilute samples with Nuclease-free Water according to the following table, dependingon the reverse transcription enzyme you are using. You need to prepare either 12 µLor 10.5 µL of each diluted sample (10 ng) for reverse transcription reactions before anIon AmpliSeq™ Chef library preparation run.

Reverse transcriptase RNA concentration Volume of RNA required perreaction

SuperScript™ IV VILO™

Master Mix0.833 ng/µL 12 µL

SuperScript™ VILO™ cDNASynthesis Kit[1]

0.95 ng/µL 10.5 µL

[1] Recommended for the Ion AmpliSeq™ Transcriptome Human Gene Expression Panel, Chef-ready Kit.

Note: If you choose to use more RNA, increase the concentration, not the volume. Forexample, to apply 100 ng of RNA per library, use 12 µL of RNA at 8.33 ng/µL, or10.5 µL of RNA at 9.5 ng/µL.

Proceed to “Reverse transcribe RNA“ on page 20, or “Reverse transcribe RNA usingthe SuperScript™ VILO™ cDNA Synthesis Kit“ on page 47 if you are using this kit.

3

Guidelines forisolating andquantifying RNA

Dilute the RNAsamples


Reverse transcribe RNA

RNA samples must be manually converted to cDNA before library preparation on theIon Chef™ Instrument. We recommend using the SuperScript™ IV VILO™ Master Mix(ordered separately), which simplifies reaction setup and provides improvedperformance.

Note:· For setting up reverse transcription reactions with the SuperScript™ III Reverse

Transcriptase, see “Reverse transcribe RNA using the SuperScript™ VILO™ cDNASynthesis Kit“ on page 47.

· Set up the reverse transcription reactions on ice or a cold block.

1. If RNA was prepared from FFPE tissue and not previously heat-treated, pre-heatat 80°C for 10 minutes, then cool to room temperature.

2. Remove, then discard the plate seal from an IonCode™ 96 Well PCR Plate.

3. Pipet 3 µL of the SuperScript™ IV VILO™ Master Mix into wells A1 to H1 of theplate.

4. Pipet 12 µL of each RNA sample (0.833 ng/µL, 10 ng total) into column 1 wellscontaining the RT Master Mix to give a 15-µL reaction volume.

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

B

C

D

E

F

G

H

21

1 Each column 1 well contains 3 µL SuperScript™ IV VILO™ Master Mix, and 12 µL ofdiluted RNA sample (0.833 ng/µL, 10 ng total), or 12 µL Nuclease-free Water as non-template control.

2 Each column 6 well contains a dried-down IonCode™ barcode. The lowest barcodenumber is in A6, and the highest is in H6. All appear light blue in the actual plates.

5. If you are processing fewer than 8 samples, it is preferable to add replicates orpositive control samples to the run. Otherwise, pipet 12 µL of Nuclease-freeWater as non-template control into column 1 wells that do not contain an RNAsample.

Note: If you are processing 5 or fewer samples, we recommend that youquantify your output combined library by qPCR to ensure that an optimalconcentration is used in templating reactions.

Chapter 3 Prepare RNA samplesReverse transcribe RNA3


6. Seal the plate with a MicroAmp™ Clear Adhesive Film, vortex thoroughly, thenbriefly centrifuge the plate to collect droplets. Alternatively, mix by pipetting atleast half the total volume up and down at least 5 times before sealing the plate.

IMPORTANT! Offset the film to the left so that the adhesive does not cover thebarcode label. If the barcode label becomes damaged, you can override the errorduring Deck Scan.

7. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermalcycler, then run the following program to synthesize cDNA.

Temperature Time

25°C 10 minutes

50°C 10 minutes

85°C 5 minutes

10°C Hold

STOPPING POINT Samples can be stored at 10°C overnight (12–16 hours) in thethermal cycler. For longer term, store at –30°C to –10°C.

8. Remove the plate from the thermal cycler, then gently tap the plate to collect anycondensate. Alternatively, centrifuge briefly to collect droplets.


Chapter 3 Prepare RNA samplesReverse transcribe RNA 3


Run the Ion Chef™ System

■ Thaw the reagents and prepare the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■ (Optional) Create a Sample Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■ Set up the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

■ Start the Ion Chef™ run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

■ Unload the Ion Chef™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

■ Clean the Ion Chef™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

■ Proceed to an Ion Chef™ template run and create a Planned Run template . . . 37

Thaw the reagents and prepare the instrument

• Before the run, thaw one Ion AmpliSeq™ Chef Reagents DL8 cartridge at roomtemperature for 20 minutes.

• If needed, thaw the Ion AmpliSeq™ primer pools.

• If not performed after a previous run, unload and clean the Ion Chef™ Instrument(see “Clean the Ion Chef™ Instrument“ on page 35).

• Confirm that the Ion Chef™ Instrument has a connection to the Torrent Server. Onthe Ion Chef™ home touchscreen, touch Settings4Torrent Server to view theconnection status of your instrument.

Note: If the instrument is not connected, see the Ion Chef™ and Torrent ServerNetwork Setup User Guide (Pub. No. MAN0013444) for instructions on how toconfigure a direct or indirect network connection of the Ion Chef™ Instrument to aTorrent Server.

(Optional) Create a Sample Set

Optional sample tracking on the Torrent Server allows you to automatically followsamples grouped in Sample Sets, from library and template preparation through chiploading, sequencing, and data analysis.

Note: Automated sample tracking from library preparation through sequencing issupported only for libraries prepared from a single Sample Set in one Ion AmpliSeq™

for Chef run. If libraries from multiple Ion AmpliSeq™ for Chef runs are super-pooledin a Library Sample Tube in an Ion Chef™ template run, you need to enter sampleinformation manually when setting up a Planned Run.

4


To create a Sample Set:

1. Log in to the Torrent Server via the Torrent Browser.

2. In the Plan tab, select Samples, then click Enter Samples Manually.

3. Click Enter New Sample.

4. In the Add Sample dialog,a. Enter a Sample Name.

b. Select the sample's PCR Plate Position (A–H).

c. Select appropriate values in the Barcode Kit (IonCode Barcodes 1−32) andBarcode fields.

d. Enter any additional information in the annotations and attributes tabs onthe right side of the dialog, then click Done.

5. Repeat steps 3 and 4 for each sample in your Sample Set (up to 8).

Chapter 4 Run the Ion Chef™ System(Optional) Create a Sample Set 4


6. Click Save Sample Set, then in the Save Samples dialog, click Add Sample Set.

7. In the Save Samples dialog, enter information or select from the availabledropdown lists:

• Sample Set Name• Group Type• Library Prep Kit• PCR Plate Serial Number (optional)• Description (optional)

Note: Give each Sample Set a unique name to avoid confusion when selecting aSample Set during creation of a Planned Run.

8. Click Save.

Chapter 4 Run the Ion Chef™ System(Optional) Create a Sample Set4


Set up the Ion Chef™ System

1. Uncap all 4 tubes in positions A, B, C, and D in the Ion AmpliSeq™ Chef ReagentsDL8 cartridge. Save the caps.

DD

AA

BB

CC

00012647

00012216

00012216

00012216

1

2

4

3

1 Position A (150 µL Primer Pool 1 at 2X concentration)2 Position B (150 µL Primer Pool 1 or 2 at 2X concentration)3 Position C (Empty tube)4 Position D (Output tube)

2. Add primer panels to the Ion AmpliSeq™ Chef Reagents DL8 cartridge using thefollowing guidelines:

Note: Use the same volumes if you are processing fewer than 8 samples.

If you areusing Action

Chef-readypanels

1. Vortex the Primer Pool Tubes to mix, then centrifuge.

2. Remove the caps, then replace the tubes in Positions A and B ofthe Ion AmpliSeq™ Chef Reagents DL8 cartridge with the Chef-ready panel tubes.

2X panels,aliquot only

Add the 2X Primer Pools to the Primer Pool Tubes.

• For a 1-pool panel, pipet 150 µL of the 2X Primer Pool into eachof the Position A and Position B tubes.

• For a 2-pool panel, pipet 150 µL of the 2X Primer Pool 1 into thePosition A tube, then pipet 150 µL of 2X Primer Pool 2 into thePosition B tube.

5X panels,dilute andaliquot

Dilute 5X primers to 2X and aliquot, or dilute 5X primers withNuclease-free Water in the Primer Pool Tubes.

• For a 1-pool panel, dilute to 2X by adding 120 µL of 5X PrimerPool to 180 µL of Nuclease-free Water. Then pipet 150 µL of the2X Primer Pool into each of the Position A and B tubes.

• For a 2-pool panel, pipet 60 µL of 5X Primer Pool 1 into thePosition A tube, and 60 µL of 5X Primer Pool 2 into the Position Btube. Then pipet 90 µL of Nuclease-free Water into each of thePosition A and Position B tubes. Using a new tip for each tube,pipet up and down 5 times to mix.

Add IonAmpliSeq™ 2XPrimer Pools toPositions A and Bof the Reagentscartridge

Chapter 4 Run the Ion Chef™ SystemSet up the Ion Chef™ System 4


Note: The tube in Position D contains 700 µL of combined barcoded libraries atcompletion of the run.

Use the following procedure to load the Ion Chef™ Instrument. A fully loaded deck isshown in the following figure:

A

B

C

D

E

F

G

H

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

1

5

7

3

2

4 6

1 Ion AmpliSeq™ Chef Solutions DL8cartridge

2 Ion AmpliSeq™ Chef Reagents DL8cartridge

3 Ion AmpliSeq™ Tip Cartridge L8

4 PCR Frame Seal5 IonCode™ 96 Well PCR Plate6 Empty Tip Cartridge L87 Enrichment Cartridge

IMPORTANT! When loading the instrument, do not force a cartridge into place. Eachcartridge fits only one location on the deck and in one orientation. If a cartridge doesnot fit, verify that you are loading the correct cartridge in the correct orientation.

1. Open the Chef door.a. On the instrument touchscreen, touch (Open Door) in the upper right

corner, then wait for the door latch to open.

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

1

1 Hold here, then lift

Load the IonChef™ Instrument

Chapter 4 Run the Ion Chef™ SystemSet up the Ion Chef™ System4


2. Load the Ion AmpliSeq™ Chef Solutions DL8 cartridge into the Solutions station.a. Gently tap the cartridge on the bench to force the reagents to the bottoms of

the tubes.

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

3. Load the Ion AmpliSeq™ Chef Reagents DL8 cartridge into the Reagents station.a. Gently tap the cartridge on the bench to force the reagents to the bottoms of

the tubes, then verify that all the liquid is at the bottom, and not splashed onthe side of the tubes.

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

IMPORTANT! Ensure that 4 flagged tubes are uncapped and loaded inPositions A–D of the Reagents cartridge, and 2X Primer Pools are loaded inPositions A and B.

4. Load an empty Tip Cartridge L8 from a previous run into the Used Pipette Tipstation.

5. Load a new Ion AmpliSeq™ Tip Cartridge L8 into the New Pipette Tip station(left side of deck).

a. Unwrap the Ion AmpliSeq™ Tip Cartridge L8, then remove the cover toexpose the pipette tips.

b. Slide the catch forward to allow the locking bracket to pivot upward. Loadthe Ion AmpliSeq™ Tip Cartridge L8 into position, pull the bracketdownward, then push the catch backward to lock the cartridge in place.

IMPORTANT! The run will fail if you do not close the locking bracket.

6. If you are preparing libraries from RNA, carefully remove the plate seal from theIonCode™ 96 Well PCR Plate containing cDNA.

IMPORTANT!· Start from the left side of the plate to minimize the chance of damaging the

barcode on the right side of the plate.· To prevent spattering of reverse transcription reactions during removal of the

plate seal, we recommend that you position the plate in a 96-well rack, thencarefully peel the seal with one hand while firmly holding the plate and rackdown on the bench with your other hand. Inspect the column 1 wells fordroplets on the side of wells. Apply a new plate seal and re-centrifuge theplate to collect droplets to the bottom of the wells, if needed.

7. Load the IonCode™ 96 Well PCR Plate containing gDNA or cDNA onto thethermal cycler sample block, with position A1 in the upper left corner, then pressdown to seat it.

8. Slide a new PCR Frame Seal underneath the automated heated cover.

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

Chapter 4 Run the Ion Chef™ SystemSet up the Ion Chef™ System 4


9. Load the Enrichment Cartridge into the Enrichment station, then press down onthe cartridge to ensure that it is level with the instrument deck.

10. Close the instrument door by first lifting it up slightly to disengage the lockingmechanism, then pushing down on the door so that the lower locks engage.

IMPORTANT! After closing the door, ensure that both sides of the door arelocked down.

Start the Ion Chef™ run

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

2. Touch Step by step, then touch AmpliSeq on the Run Options screen.

Note: To bypass the step by step deck loading guide, touch Quick start.

3. Ensure that you have completely loaded the Ion Chef™ deck with Ion AmpliSeq™

Kit for Chef DL8 consumables by advancing through the Step by Step deckloading steps on the instrument touch screen.

Chapter 4 Run the Ion Chef™ SystemStart the Ion Chef™ run4


4. Touch Start check on the Close Door screen. The Ion Chef™ Instrument performsa Deck Scan.

Note: If the PCR plate is not recognized, select the appropriate plate whenprompted. If no sample set was selected or planned in the Torrent Server, thefollowing warning appears: "No sample Set detected. Do you want to continue?"Touch OK.

5. After Deck Scan completes (~3 minutes), touch Next.

6. On the Data Destination screen, verify the Server and Sample set information,then touch Next.

Chapter 4 Run the Ion Chef™ SystemStart the Ion Chef™ run 4


7. Enter the appropriate number of primer pools, target amplification cycles, and ananneal/extension time for your run.

Recommended number of amplification cycles

Primer pairs perpool

(excluding genefusion primer

pairs)[1]

Recommended number of amplificationcycles

(10 ng DNA/RNA, 3,000 copies)[2]

Anneal/Extensiontime

High qualityDNA/RNA

Low qualityDNA/RNA

(FFPE DNA/RNA orcfDNA/RNA)

Panels for genefusion detection

only

28 31 4 minutes

12–24 22 25 4 minutes

25–48 21 24 4 minutes

49–96 20 23 4 minutes

97–192 19 22 4 minutes

193–384 18 21 4 minutes

385–768 17 20 4 minutes

769–1,536 16 19 8 minutes

1,537–3,072 15 18 8 minutes

3,073–6,144 14 17 16 minutes

6,145–24,576 13 16 16 minutes

[1] When using RNA panels with a mix of primer pairs for gene expression and gene fusion detection, use the number of gene expression primer pairs only to determine the appropriate number of amplification cycles.

[2] Source: Ion AmpliSeq™ Library Preparation User Guide (Pub. No. MAN0006735). Because nucleic acid is divided between two amplification reactions on the Ion Chef™ Instrument, one additional cycle is recommended above the standard manual Ion AmpliSeq™ protocol. Additional cycles can be added if sample quantity or quality is questionable.



Cycle number that is recommended in the preceding table is based on 10 ngDNA/RNA input. Adjust cycle number for lower or higher DNA/RNA input:

Amount of DNA/RNA starting material Adjustment to cycle number

1 ng (300 copies) +3

10 ng (3,000 copies) 0

100 ng (30,000 copies) –3

Exceptions to the amplification parameters recommended in the parameter table

Ion AmpliSeq™ panel Primerpairs/pool Description of change

Ion AmpliSeq™

Pharmacogenomics ResearchPanel, Chef-ready (Cat. No.A29998)

119 Add 2 amplification cycles (21cycles for high-quality DNA, 24cycles for FFPE DNA)

Ion AmpliSeq™ TranscriptomeHuman Gene Expression Panel,Chef-ready (Cat. No. A31446)

20,800 • Use 12 cycles for high-quality RNA

• Use 17 amplification cyclesfor FFPE RNA instead of 16cycles

Ion AmpliSeq™ panels using a375-bp amplicon design

— Add 4 minutes to theanneal/extend timerecommended in the table

8. Touch Start Run.

9. After approximately 7 hours, return to the Ion Chef™ Instrument. On the RunComplete screen, touch Next to proceed to the unloading and cleaning steps.

IMPORTANT! After a run, the Ion Chef™ Instrument holds the barcoded librariesin the tube that is loaded in Position D of the Reagents cartridge. To avoid fluidloss due to evaporation, remove and cap the tube of combined barcoded librariesas soon as possible after run completion. Do not leave the tube in the instrumentlonger than 24 hours after the start of the run. After 24 hours from the start of therun, the instrument chiller stops actively cooling, and the sample is held at 27°C.

Chapter 4 Run the Ion Chef™ SystemStart the Ion Chef™ run 4


Note: Touch Run Details to view run information.

You can also monitor the run on the Torrent Browser by selecting Monitor4IonChef, and viewing the Library Prep Progress bar and Library Prep Statusassociated with your Sample Set.



Unload the Ion Chef™ Instrument

Remove the tube containing the combined library at approximately 100-pMconcentration from Position D of the Reagents cartridge, and remove usedconsumables from the instrument from the indicated stations.

1

4

3

2

5

6

1 Solutions station2 Reagents station3 New Pipette Tip station: move the empty Tip Cartridge to the Used Pipette Tip station4 Thermal cycler sample block5 Used Pipette Tip station6 Enrichment station

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

to open.

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

1

1 Hold here, then lift

2. Remove the Ion AmpliSeq™ Chef Reagents DL8 cartridge. Remove and cap thecombined library tube from Position D, then discard the cartridge.

3. Remove, then discard the Ion AmpliSeq™ Chef Solutions DL8 cartridge.

Chapter 4 Run the Ion Chef™ SystemUnload the Ion Chef™ Instrument 4


4. Remove, then discard the IonCode™ 96 Well PCR Plate and seal from the thermalcycler sample block.

5. Remove, then discard the box ofused pipette tips from the UsedPipette Tip station.

Note:

· Handle the disposable reservoir in the Used Tip station with care. During therun, liquid waste collects in the reservoir.

· Appropriately discard the liquid waste by tipping the reservoir on the cornerwith the slot and pouring the waste into a waste container through that slot.

Note: Do not reuse the Used pipette tip rack.

6. Move the empty Tip Cartridge L8 from the New Pipette Tip station to the UsedPipette Tip station.

IMPORTANT! Do not discard the empty Ion AmpliSeq™ Tip Cartridge L8.

7. Remove and discard the Enrichment Cartridge.

The libraries are at ~100 pM (total combined library concentration) and are ready touse in template preparation. Store unused portions of combined libraries at 4°C to 8°Cfor up to 1 month. For longer-term storage, store at –30°C to –10°C. See theappropriate Ion Chef™ or Ion OneTouch™ 2 template kit user guide for detailedinstructions for template preparation.

To combine Ion AmpliSeq™ libraries from multiple Ion Chef™ runs, see Appendix C,“Strategies for combining Ion AmpliSeq™ libraries“.

(Optional)Combine librariesfrom multipleIon Chef™ runs

Chapter 4 Run the Ion Chef™ SystemUnload the Ion Chef™ Instrument4


Clean the Ion Chef™ Instrument

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 theIon Chef™ Instrument, make certain to observe sterile laboratory procedures at alltimes to ensure minimal contamination.

IMPORTANT! Clean the Ion Chef™ Instrument after every run. To preventcontamination, do not operate the instrument unless it has been recently cleaned.

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

2. On the Ion Chef™ Instrument touchscreen that appears after run completion,touch Next.

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

About the cleaningprotocol

Clean theIon Chef™

Instrument

Chapter 4 Run the Ion Chef™ SystemClean the Ion Chef™ Instrument 4


3. Ensure that you have removed all consumables from the Ion Chef™ Instrument,then touch Next.

4. With the door closed, touch Start.

The instrument performs a Deck Scan before starting the cleaning routine. TheIon Chef™ Instrument stops ventilation and illuminates the ultraviolet (UV) lightin the instrument.

CAUTION! The Ion Chef™ Instrument emits UV light at 254 nm. Wearappropriate eye wear, protective clothing, and gloves when working near theinstrument. Do not look directly at the UV light while it is illuminated duringthe cleaning routine.

Chapter 4 Run the Ion Chef™ SystemClean the Ion Chef™ Instrument4


Proceed to an Ion Chef™ template run and create a Planned Runtemplate

If you are using the Ion Chef™ Instrument to prepare template-positive ISPs and loadIon sequencing chips, you can use the sample-tracking capability of the system totransfer information from your Sample Set to your Planned Run automatically. Whenyour Ion AmpliSeq™ run is complete, check the status of your run on the TorrentBrowser.

1. In the Plan tab, click Samples, then find your Sample Set in the list.

Status of Ion AmpliSeq™ for Chef Sample Sets, listed on the far right column, canbe one of three types:

Status Indication

libPrep_pending New Sample Set.

libPrep_reserved The Sample Set is currently running on Ion Chef.You can monitor status on the Monitor page.

libPrep_done The Ion Chef™ Instrument has finished the libraryprep run. Ready for template run.

2. If the status of your Sample Set islibPrep_done, click Settings aligned with your Sample Set, thenselect Plan Run.

Chapter 4 Run the Ion Chef™ SystemProceed to an Ion Chef™ template run and create a Planned Run template 4


3. In the dialog that appears, select an existing Run Template populated withpreviously saved parameters from the dropdown list:

Note: To start from a new Run Template, select Add new template:

4. Select or change settings appropriate to your Planned Run in the Create Templatewizard.

Chapter 4 Run the Ion Chef™ SystemProceed to an Ion Chef™ template run and create a Planned Run template4


5. Click Save Template in the Save tab. The Torrent Browser then opens theBarcoding tab, where your Sample Set information is displayed.

Chapter 4 Run the Ion Chef™ SystemProceed to an Ion Chef™ template run and create a Planned Run template 4


6. In the Sample Tube Label: field, enter or scan the Sample Tube Label from theLibrary Sample Tube in the Reagents cartridge. Your Planned Run is loadedwhen the Ion Chef™ Instrument performs a Deck Scan during the templatepreparation run.

Note:· If you do not enter the Sample Tube Label, you are prompted to select a

Planned Run manually using the Ion Chef™ screen interface.· The combined library tube label from library preparation is stored with the

sample set for archiving convenience and is associated with the Planned Runin automated sample tracking.

· Automated sample tracking from library preparation to template preparationand sequencing is supported only for libraries that are prepared from oneSample Set in one Ion AmpliSeq™ for Chef run. If libraries from multiple IonAmpliSeq™ for Chef runs are super-pooled in a Library Sample Tube in anIon Chef™ template run, you need to enter sample information manually whensetting up a Planned Run.

7. Click Next twice to proceed to the Save and Finish tab, enter a name for yourPlanned Run, then click Save and Finish.

Chapter 4 Run the Ion Chef™ SystemProceed to an Ion Chef™ template run and create a Planned Run template4


Tips and troubleshooting

Tips

• Up to 100 ng of DNA or RNA can be used in the standard protocol if the samplequality or quantity is variable. Subtract up to 3 cycles from the targetamplification if uniformity suffers.

• When using the Ion AmpliSeq™ Direct FFPE DNA Kit, samples may be processedin Eppendorf™ DNA LoBind™ Microcentrifuge Tubes before target amplification.

• When using the Ion AmpliSeq™ Comprehensive Cancer Panel, the 4 primer poolsprovided can be collapsed into 2 primer pools for library preparation on theIon Chef™ System. Use the following procedure:

a. Mix equal volumes of Pool 1 and Pool 2 (new Pool 1).b. Mix equal volumes of Pool 3 and Pool 4 (new Pool 2).c. Adjust target amplification cycle number and anneal/extend time

accordingly for the 2 new pools (~8,000 primer pairs/pool).

Note: 2-3% of amplicons may be compromised due to overlap.

A


Ion AmpliSeq™ Direct FFPE DNA Kit

Observation Possible cause Recommended action

Tissue does not transfer fromslide

Not enough Transfer Solution. Hold the slide at a 45° angle and pipet extraTransfer Solution to the top of the slideallowing the tissue to flow towards the bottom.Remove collected tissue from the bottom,repeat as needed.

Tissue is clumpy. Transfer the mass of tissue to a collectiontube, then continue breaking it up with a pipettip.

Pre-incubate with Transfer Solution on slidefor 5 minutes, then proceed to scraping.

Difficulty scraping tissue off theslide

Tissue is fibrous. Scrape with 200‑µL tip prior to transfer, using acircular motion, then continue with a 20‑µL tip.

Scrape and homogenize tissue with a scalpelblade, then continue breaking up tissue with a20‑µL tip.

Repeat transfer process with a larger volumeof Transfer Solution.

Target tissue area issurrounded by undesiredtissue.

Use a scalpel blade to scrape away undesiredtissue or paraffin, then use Transfer Solution tocollect the desired tissue.

Excess undissolved tissue inDirect Reagent

Too much tissue in reaction. Use 4– 100 mm2 tissue section. Tissue sectionsshould be 5–10 µm thick.

Digest may be incomplete. Incubate for an additional 5–15 minutes at65°C. After digestion sample may still becloudy, this will not affect performance. Ensurehomogenous mixing of the sample prior toremoving an aliquot for target amplification.Undissolved tissue that can be aspirated with apipet tip may still be added to the TargetAmplification reaction.

Centrifuge at ≥1,000 × g for 1 minute, thentransfer 15 µL to a fresh tube, avoiding thefibrous pellet.

Transfer Solution and DirectReagent do not separate intotwo phases

Too much paraffin in sample. Use a scalpel blade to scrape away undesiredparaffin prior to adding Transfer Solution to thedesired tissue area.

Centrifuge at ≥1,000 × g for 1 minute, thentransfer 15 µL to a fresh tube, avoiding thefibrous pellet and tube walls.

Appendix A Tips and troubleshootingIon AmpliSeq™ Direct FFPE DNA KitA



Transfer Solution and DirectReagent do not separate intotwo phases

Perform partial deparaffinization before addingTransfer Solution to tissue on the slide:

1. Submerge slide in 100% xylene for30 seconds.

2. Remove the slide, then drain any excessxylene.

3. Submerge slide in 100% ethanol for30 seconds.

4. Remove the slide, then allow to air dry.

Difficulty transferring lower(aqueous) phase to targetamplification reaction

Transfer Solution is in pipet tip. Return tip contents to reaction tube, thencentrifuge at ≥1,000 × g for 1 minute toseparate phases. Move pipet quickly throughthe upper phase when transferring.

Note: Transfer Solution will not interfere withtarget amplification.

Low AmpliSeq™ libraryconcentration

Insufficient tissue was used. Use 25–100 mm2 tissue section of 5−10 µmthickness. If needed, use multiple slides toobtain 25–100 mm2 tissue.

Insufficient amplifiable DNAwas used.

FFPE DNA quality may vary due to tissuefixation methods, length of storage time, andstorage conditions. Although the Qubit™ assaymay detect the presence of DNA, the DNA maynot be of sufficient quality to generate anAmpliSeq™ library. Re‑prepare FFPE DNA from100 mm2 tissue section of 5−10 µm thickness.If needed, use multiple slides to obtain100 mm2 tissue.

Inhibitors are present in thetissue.

Inhibitors such as high melanin content canaffect PCR, reduce the volume of input samplegoing into the target amplification reaction.

Qubit™ result indicates highconcentration (>10 ng/µL)

FFPE tissue has high DNAcontent.

Reduce the volume of input sample going intothe target amplification reaction by one half toone quarter.

Qubit™ result indicates lowconcentration (<0.5 ng/µL)

Samples with low DNA yieldcan still be sufficient togenerate an AmpliSeq™ library.

FFPE tissue has low DNAcontent.

Increase the number of target amplificationcycles by 2 or 3.

Appendix A Tips and troubleshootingIon AmpliSeq™ Direct FFPE DNA Kit A


Ion AmpliSeq™ library troubleshooting


Chip loading or library yield islow(Library yield checked by qPCRusing the Ion Library TaqMan®

Quantitation Kit, Cat. No.4468802.)

Input DNA or RNA was mis-quantified.

Re-quantify input DNA using the TaqMan®

RNase P Detection Reagents Kit; quantify RNAwith the Qubit™ RNA HS Assay Kit using theQubit™ 3.0 Fluorometer.

Less than 10 ng of inputDNA/RNA was loaded.

Add more DNA/RNA by increasing theconcentration, or increase target amplificationcycles.

Double the amount of library added to thetemplate preparation reaction.

Uniformity is poor, AT-richamplicons are under-represented

PCR was inefficient. Double the anneal and extend time.

Long amplicons are under-represented (short libraryreads)

Sample DNA or RNA isdegraded.

Use an FFPE assay design for degraded or lowquality samples.

PCR was inefficient. Double the anneal and extend time.

Too few nucleotide flows wereused.

Use an appropriate number of flows tosequence through amplicons.

The number of on-target readsis lower than expected

Unknown. Increase the number of target amplificationcycles by two.

Sample ID Panel targets arecounted as off-target reads.

Add back the on-target reads from the SampleID Panel.

Percentage of polyclonal ISPs ishigh (>40%)

Library input was too high. Decrease the amount of library added to thetemplate preparation reaction by 50%.

Re-quantify with the Ion Library TaqMan®

Quantitation Kit to confirm concentration, thendilute if needed.

Other. Check the appropriate template preparationuser guide for more information.

Low quality ISPs are present athigh percentage (> 15%)

Library input was too low. Double the volume of library used in templatepreparation.

Use a fresh dilution of library prepared in alow-bind tube.

Other. Check the appropriate template preparationuser guide for more information.

Appendix A Tips and troubleshootingIon AmpliSeq™ library troubleshootingA


Ion Chef™ System setup and operation


Instrument does not display thehome screen when powered on

Multiple causes are possible. 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, thenoperate the instrument normally.

A hardware or software errorhas occurred.

Contact Technical Support to report theproblem and for further assistance.

Instrument will not start 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 one or more errorsdisplayed, replace the missingconsumable as directed, press No whenprompted, then press Next to cancel therun. After returning to the home screen,restart the run.

2. If the error persists, ensure that:

• All cartridges and PCR plate areloaded correctly and are level on theinstrument deck.

• The barcodes of the 4 flagged tubesin Positions A–D of the Reagentscartridge are visible and positionedcorrectly.

• All tubes are both present anduncapped in the Reagents cartridge.

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

• If you are confident that theIon Chef™ Instrument is set upcorrectly, and you are comfortabledisregarding the warnings, touch YESfollowing Deck Scan to proceed withthe run.

• If the instrument cannot start therun, contact Technical Support forfurther help.

The instrument hasencountered an internal error.

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

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

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.

Appendix A Tips and troubleshootingIon Chef™ System setup and operation A



Instrument stops during a run A consumable was improperlyloaded.

Ensure that all consumables are loadedaccording to the instructions provided.

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 network connection.

• 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.

Appendix A Tips and troubleshootingIon Chef™ System setup and operationA


Supplemental procedures

■ Reverse transcribe RNA using the SuperScript™ VILO™ cDNASynthesis Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

■ (Optional) Remove deaminated bases from Direct FFPE DNA . . . . . . . . . . . . . . 49

■ (Optional) Qubit™ Fluorometer: Quantify the FFPE DNA . . . . . . . . . . . . . . . . . . 49

Reverse transcribe RNA using the SuperScript™ VILO™ cDNASynthesis Kit

Reverse transcription reactions can be performed with the SuperScript™ VILO™ cDNASynthesis Kit, which contains the 10X SuperScript™ III Enzyme Mix. This enzyme mixrequires that you prepare a reverse transcription Master Mix with 5X VILO™ RTReaction Mix before use. Follow the procedure below.

Note: Set up the reverse transcription reaction on ice or a cold block.

1. If RNA was prepared from FFPE tissue and not previously heat-treated, pre-heatat 80°C for 10 minutes, then cool to room temperature.

2. Prepare a reverse transcription Master Mix. For 8 samples, combine the followingcomponents in a 1.5-mL tube. Vortex for 1−3 seconds to mix, then centrifuge.Adjust volumes appropriately for fewer samples.

Component Volume per reaction Volume for 9 reactions[1]

5X VILO™ RT Reaction Mix(blue cap)

3 µL 27 µL

10X SuperScript™ IIIEnzyme Mix (black cap)

1.5 µL 13.5 µL

[1] One reaction added as overage to compensate for pipetting error.

IMPORTANT! If there is visible precipitate in the 5X VILO™ RT Reaction Mix,vortex or pipet up and down to resuspend. Ensure that there is no precipitate inthe tube cap.

3. Remove, then discard the plate seal from an IonCode™ 96 Well PCR Plate.

4. Pipet 4.5 µL of the reverse transcription Master Mix into wells A1 to H1 of theplate.

B


5. Pipet 10.5 µL of each RNA sample (0.95 ng/µL, 10 ng total) into column 1 wellscontaining the RT Master Mix to give a 15-µL reaction volume.

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

B

C

D

E

F

G

H

21

1 Each column 1 well contains 4.5 µL reverse transcription Master Mix, and 10.5 µL ofdiluted RNA sample (0.95 ng/µL, 10 ng total).

2 Each column 6 well contains a dried-down IonCode™ barcode. The lowest barcodenumber is in A6, and the highest is in H6. All appear light blue in the actual plates.

6. If you are processing fewer than 8 samples, it is preferable to add replicates orpositive control samples to the run. Otherwise, pipet 10.5 µL of Nuclease-freeWater into column 1 wells that do not contain an RNA sample.

Note: If you are processing 5 or fewer samples, we recommend that youquantify your output combined library by qPCR to ensure that an optimalconcentration is used in templating reactions.

7. Seal the plate with a MicroAmp™ Clear Adhesive Film, vortex thoroughly, thencentrifuge briefly to collect droplets. Alternatively, mix by pipetting at least halfthe total volume up and down at least 5 times before sealing the plate.

IMPORTANT! Offset the film to the left so that the adhesive does not cover thebarcode label. If the barcode label becomes damaged, you can override the errorduring Deck Scan.

8. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermalcycler, then run the following program to synthesize cDNA.

Temperature Time

42°C 30 minutes

85°C 5 minutes

10°C Hold

STOPPING POINT Samples can be stored at 10°C overnight (12–16 hours) in thethermal cycler. For longer term, store at –30°C to –10°C.

9. Remove the plate from the thermal cycler, then gently tap the plate to collect anycondensate. Alternatively, centrifuge briefly to collect droplets.


Appendix B Supplemental proceduresReverse transcribe RNA using the SuperScript™ VILO™ cDNA Synthesis KitB


(Optional) Remove deaminated bases from Direct FFPE DNA

FFPE preservation methods can lead to significant cytosine deamination within theisolated DNA, and result in decreased sequencing quality. When using the IonAmpliSeq™ Direct FFPE DNA Kit, deaminated cytosines (uracil) can be enzymaticallyremoved by treatment with Uracil DNA Glycosylase (UDG) prior to targetamplification.

1. Add 1–2 units of UDG to the Direct FFPE DNA aqueous lower phase of eachsample after the 15-minute incubation at 65°C.

2. Set a 20-µL pipette to 15 µL, then mix the lower phase by pipetting up and down10 times.

3. (Optional) If necessary, centrifuge briefly to collect contents and re-separate theliquid phases in the bottom of the tube.

4. Seal the plate, then incubate at 37°C for 5 minutes, followed by 5 minutes at 65°C.

Proceed immediately to “Remove an aliquot for library preparation“ on page 17.

(Optional) Qubit™ Fluorometer: Quantify the FFPE DNA

When using the Ion AmpliSeq™ Direct FFPE DNA Kit, the DNA concentration can beestimated using the Qubit™ 2.0 or Qubit™ 3.0 Fluorometer and the Qubit™ dsDNA HSAssay Kit (Cat. No. Q32851). See the Qubit™ dsDNA HS Assay Kits User Guide(Pub. No. MAN0002326) for more information.

1. Set up the required number of 0.5-mL Qubit™ Assay tubes for standards andsamples. The Qubit™ dsDNA HS Assay requires 2 standards.

2. Prepare sufficient Qubit™ working solution for all samples and standards bydiluting Qubit™ dsDNA HS Reagent 1:200 in Qubit™ dsDNA HS Buffer.

3. Combine 2 µL of the FFPE DNA sample with 198 µL (200-µL final volume) ofworking solution, mix well, then incubate for at least 2 minutes.

4. Prepare each Qubit™ standard as directed in the user guide.

5. Measure the concentration of each sample and standard on the Qubit™

Fluorometer.

6. Calculate the concentration of the undiluted sample by multiplying by thedilution factor.

Proceed to “Dilute the DNA samples“ on page 17.

Appendix B Supplemental procedures(Optional) Remove deaminated bases from Direct FFPE DNA B


Strategies for combining IonAmpliSeq™ libraries

■ Combine barcoded libraries from multiple runs for sequencing on asingle Ion chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

■ Combine libraries prepared using different panels for equal coverage . . . . . . . 51

■ Combine unequal volumes of libraries for varying depth of coverage . . . . . . . . 52

■ Ion Chip capacities for Ion AmpliSeq™ DNA libraries sequenced atequal depths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

■ Ion Chip capacities for Ion AmpliSeq™ RNA libraries . . . . . . . . . . . . . . . . . . . . . 54

This section describes various strategies for super-pooling Ion AmpliSeq™ librariesprepared in multiple runs of the Ion Chef™ Instrument.

Combine barcoded libraries from multiple runs for sequencing on asingle Ion chip

The Ion AmpliSeq™ Kit for Chef DL8 contains reagents and materials sufficient toprepare up to 32 uniquely barcoded libraries. If the same Ion AmpliSeq™ panel (ortwo panels with the same number of amplicons) was used over multiple runs withdifferent barcodes, the combined libraries can be super-pooled by adding equalvolumes of each. See “Ion Chip capacities for Ion AmpliSeq™ DNA librariessequenced at equal depths“ on page 53 and “Ion Chip capacities for Ion AmpliSeq™

RNA libraries“ on page 54 to determine how many libraries you can sequence onvarious chips, given the number of primer pairs in the Ion AmpliSeq™ panel you used,and the coverage depth you require.

Note: Optional automated sample tracking is not supported if combined librariesprepared in multiple Ion AmpliSeq™ for Chef runs are super-pooled.

C


Combine libraries prepared using different panels for equalcoverage

Use this method to combine libraries from different panels to be sequenced on asingle chip at approximately the same depth. To prepare 100 µL of combined libraryfrom libraries prepared from two different panels:

1. Dilute each library to ~100-pM concentration.

2. Combine the libraries using the following formula:

● Volume (μL) of library from Panel 1 = 100 μL Number of primer pairs in Panel 1

Total number of primer pairs in Panels 1 and 2

● Volume (μL) of library from Panel 2 = 100 μL Number of primer pairs in Panel 2

Total number of primer pairs in Panels 1 and 2

Example:Number of primer pairs in Panel 1 = 207Number of primer pairs in Panel 2 = 20Volume of library from Panel 1 = 100 µL × (207/227) = 91 µLVolume of library from Panel 2 = 100 µL × (20/227) = 9 µLTotal volume of combined libraries = 100 µL

Appendix C Strategies for combining Ion AmpliSeq™ librariesCombine libraries prepared using different panels for equal coverage C


Combine unequal volumes of libraries for varying depth of coverage

Ion AmpliSeq™ libraries with unique barcodes may be super-pooled to producedisproportionate numbers of reads. Use this strategy for combining DNA and RNAlibraries to adjust the number of reads as desired.

For example, when comparing tumor and normal sample pairs with the same panel,an average depth of coverage at ~250X may be preferred for the normal sample, whilean average depth of coverage at ~2500X may be preferred for the tumor sample. Inthis case, 8 barcoded normal and 8 tumor libraries can be super-pooled at a 1:9(normal:tumor) ratio by adding 10 µL of combined library from the normal samplerun with 90 µL of the combined library from a tumor sample run.

Sample Fractionalvolume/reads

Combined librariesfrom Chef run #1(normal)

0.1

Combined librariesfrom Chef run #2(tumor)

0.9

Sum 1.0

Appendix C Strategies for combining Ion AmpliSeq™ librariesCombine unequal volumes of libraries for varying depth of coverageC


Ion Chip capacities for Ion AmpliSeq™ DNA libraries sequenced atequal depths

The number of combined libraries that can be accommodated in a single sequencingrun depends on the chip, the balance of barcoded library concentration, and thecoverage required.

For a given chip, as the number of amplicons increases, the number of libraries thatcan be accommodated per sequencing run decreases. This relationship is shown in thefollowing table. The numbers in the following table serve as a guide for approximatecapacities. As the number of libraries per chip increases, it becomes more difficult tobalance the reads between libraries. In addition, libraries from FFPE tissue tend toproduce more variable results. We suggest combining fewer libraries initially anddetermining real limits empirically.

Ion 314™ Chip Ion 316™ Chip Ion 318™ Chip/Ion 520™ Chip Ion 530™ Chip Ion PI™ Chip/

Ion 540™ Chip

Average depth 150X 2500X 150X 2500X 150X 2500X 150X 2500X 150X 2500X

Expected coverage 95%>30X

95%>500X

95%>30X

95%>500X

95%>30X

95%>500X

95%>30X

95%>500X

95%>30X

95%>500X

Number ofamplicons per

libraryApproximate number of libraries per chip

12–24 84 8 272 26 >384 48 >384 152 >384 >384

25–48 48 4 152 14 272 26 >384 84 >384 272

49–96 26 2 84 8 152 14 >384 48 >384 152

97–192 14 1 48 4 84 8 272 26 >384 84

193–384 8 — 26 2 48 4 152 14 >384 48

385–768 4 — 14 1 26 2 84 8 272 26

769–1,536 2 — 8 — 14 1 48 4 152 14

1,537–3,072 1 — 4 — 8 — 26 2 84 8

3,073–6,144 — — 2 — 4 — 14 1 48 4

6,145–12,288 — — 1 — 2 — 8 — 26 2

12,289–24,576 — — — — 1 — 4 — 14 1

24,577–49,152 — — — — — — 2 — 8 —

Appendix C Strategies for combining Ion AmpliSeq™ librariesIon Chip capacities for Ion AmpliSeq™ DNA libraries sequenced at equal depths C


Ion Chip capacities for Ion AmpliSeq™ RNA libraries

We recommend that you plan for an average of 5,000 reads per amplicon for an IonAmpliSeq™ RNA library targeting 1– 200 genes. The actual sequencing depth requireddepends on the expression levels of the gene targets in your sample RNA, so scale thesequencing depth to accommodate your sample type and research needs.

For panels containing fusion detection primer pairs, higher library multiplexing ispossible as most targets will not be present, and therefore will not create librarymolecules. For most fusion detection assays, only ~250,000 reads/library are required.

Use the following formula and chip capacity table to provide initial guidance formultiplexing RNA-derived gene expression libraries on Ion sequencing chips.

Number of libraries sequenced per chip = Chip capacity in reads

(Sequencing depth) (Number of primer pairs)

These recommendations serve as suggestions only and the actual capacity tomultiplex libraries is determined by the expression levels of the genes included inyour Ion AmpliSeq™ RNA panel. The expression levels of the individual genes canvary by input RNA type as well. We suggest using the formula for new panels anddetermining actual multiplexing limits empirically.

Ion Chip

Ion 314™

ChipIon 316™

Chip

Ion 318™/Ion 520™

Chip

Ion 530™

Chip

Ion PI™/Ion 540™

Chip

Chip capacityin reads (M)

0.3−0.5 1−2 3−5 15−20 60−80

Example:

Chip capacity of Ion 540™ Chip = ~60,000,000 reads

Sequencing depth desired = 5000 reads per amplicon

Number of primer pairs = 100

60,000,000 / (5,000 × 100) = 120 libraries per Ion 540™ Chip

Appendix C Strategies for combining Ion AmpliSeq™ librariesIon Chip capacities for Ion AmpliSeq™ RNA librariesC


Ion AmpliSeq™ Made-to-OrderPanels

Each Ion AmpliSeq™ Made-to-Order Panel order includes primer pools at thestandard 2X or 5X concentration, in some cases, 384-well plates containing all theindividual primer pairs. Each plate well contains the forward and reverse primer inLow TE at a concentration of 307 µM per primer. You can use the plates to:

• remake the entire panel• remake a smaller panel containing a subset of primer pairs• combine two panels• make a 50X spike-in panel to add to other panels

Primer pairs from different panels can be combined as long as the amplicons within apool do not overlap. When a panel with a new footprint is created, the designed BEDfile should be modified accordingly.

Prepare primer pools from plates

To create a primer pool from 384-well plates, combine and dilute the desired primerpairs to the appropriate concentration:

• For panels with up to 96 primer pairs per pool, combine and dilute to 1,000 nM(5X, 1,000 nM).

• For panels with 97–1,228 primer pairs per pool, combine and dilute to 250 nM(5X, 250 nM).

• For panels with 1,229–3,072 primer pairs per pool, combine and dilute to 100 nM(2X, 100 nM).

D


For example, to create a primer pool with 2 µL of each primer pair:

1. Vortex the sealed primer plate, then centrifuge briefly to collect contents.

2. Remove the plate seal, then transfer 2 µL of each desired primer pair into a tube.

3. Add Low TE (10 mM Tris, pH 8.0; 0.1 mM EDTA) to the tube to the appropriatefinal volume shown in the following table:

To make primer pools Action

5X, 1,000 nM(12−96 primer pairs)

Add Low TE to a final volume of 0.614 mL[1]

5X, 250 nM(97−1,228 primer pairs)

Add Low TE to a final volume of 2.456 mL [2]

2X, 100 nM(1,229−3,072 primer pairs)

Add Low TE to a final volume of 6.140 mL

50X spike-in panel, 2,500 nM(1−123 primer pairs)

Add Low TE to a final volume of 0.246 mL

[1] Can be prepared at 2X, 400 nM by increasing the final volume to 1.536 mL.[2] Can be prepared at 2X, 100 nM by increasing the final volume to 6.140 mL.

4. Mix thoroughly by vortexing, then centrifuge briefly to collect contents.The primer pools are ready to use.

Appendix D Ion AmpliSeq™ Made-to-Order PanelsPrepare primer pools from platesD


Ion AmpliSeq™ Sample ID Panel

Using the Sample ID Panel

If you are using the Ion AmpliSeq™ Kit for Chef DL8, use the following procedure tocreate a sample signature in your panel.

1. Add 15 µL of the Ion AmpliSeq™ Sample ID Panel and 135 µL of the primerpanel to one or both Primer Pool Tubes.

Note: The Ion AmpliSeq™ Sample ID Panel can be used to match a tumor andnormal sample. However, copy number variations in the tumor sample candistort the allele balance in the fingerprint.

2. Select the following settings when creating a new Planned Run in the TorrentBrowser Planned Run Wizard:

a. In the Kits screen, select the Control Sequence, then choose Ion AmpliSeqSample ID panel from the dropdown list.

b. In the Plugins screen, select the coverageAnalysis plugin checkbox, thenclick Configure (see “Configure and run the Torrent Coverage AnalysisPlugin“ on page 59).

c. In the configuration dialog, select the Sample Tracking checkbox.

Note: Selecting the Sample Tracking checkbox enables the analysis toproduce a statistic for reads mapped to Sample ID targets so that the level ofoff-target reads is accurately represented in the Coverage Analysis Report. IfSample Tracking is not selected, Sample ID reads are counted as off-targetreads.

d. In the Plugins screen, select the sampleID plugin. A Sample ID Report isthen automatically generated after the run.

E


3. Following sequencing, select the Data tab in the Torrent Browser, then selectCompleted Runs and Results. Open the report for the run, then scroll down tothe Plugin Summary section to find the sampleID plugin results.

Appendix E Ion AmpliSeq™ Sample ID PanelUsing the Sample ID PanelE


Data analysis

For detailed information for setting up and performing data analysis with TorrentSuite™ Software, see Torrent Suite™ Software Help accessed from the Torrent BrowserHelp menu, or downloaded from thermofisher.com as a PDF (Pub. No.MAN0014668). Here you will find information for run planning, data reporting,plugin configuration, variant calling, reference file management, and other topics.

See the Ion Reporter™ Software Help (Pub. No. MAN0013516) for detailed informationfor using the suite of bioinformatics tools in Ion Reporter™ Software to streamlinevariant analysis and reporting.

The following is a brief description of configuring and running the Torrent CoverageAnalysis and Torrent ampliSeqRNA Plugins.

Torrent Coverage Analysis Plugin

The Torrent Coverage Analysis Plugin provides statistics and graphs describing thelevel of sequence coverage produced for targeted genomic regions.

There are two ways to run the Torrent Coverage Analysis Plugin:• Automatically, by selecting and configuring the plugin during run planning or• Manually, allowing you to run the plugin at any time from a completed run

report.

1. To run the Coverage Analysis Plugin automatically during run analysis, selectthe coverageAnalysis checkbox in the Plugins screen in the template or PlannedRun Wizard. After selecting the checkbox, a Configure link appears:

F

Configure and runthe TorrentCoverage AnalysisPlugin


https://www.thermofisher.com

2. Click Configure, select the settings appropriate to your run, then click SaveChanges.

The run report includes basic run metrics, but more detail can be found by clickingthe coverageAnalysis.html link, including options to download a barcode summaryreport, and an amplicon coverage matrix.

Coverage AnalysisReport

Appendix F Data analysisTorrent Coverage Analysis PluginF


More detailed information can be obtained for each barcoded library by clicking theBarcode ID.

At the top of the Coverage Analysis Report is a table that shows amplicon statistics onthe left side and base statistics on the right side. All metrics are defined with pop-upexplanations. The plugin run options can be reviewed as pop-up help on the reporttitle "Coverage Analysis Report".

The Representation Plots (which are revealed by clicking the triangle in the blue bar)allow you to visualize amplicon representation by GC content and length. If thedesigned BED file defines multiple primer pools, a mean target depth per primer poolrepresentation plot is also available.

Appendix F Data analysisTorrent Coverage Analysis Plugin F


In the following example, pool 1 is under-represented:

The Amplicon Coverage Chart shows amplicons binned by representation, low tohigh, with various overlays. Zoom into this graph to see how many amplicons are ineach bin, and click a bar to get more information on that group of amplicons.

By clicking the magnifying glass in the upper right corner of the Amplicon CoverageChart, you can change and filter what is shown on the chart by number of reads,chromosome, or gene.



The Reference Coverage Chart shows the strand-specific coverage in red and green foreach amplicon. The following example shows the coverage for amplicons in the TP53gene:

At the bottom of the Coverage Analysis Report, you can click links to download thedata in this report and the BAM and BAI files that are required for IGV (IntegratedGenomics Viewer). Read numbers that are reported in the amplicon coveragesummary file can be used to estimate RNA expression levels in Ion AmpliSeq™ RNAlibraries.

Appendix F Data analysisTorrent Coverage Analysis Plugin F


The following figure shows an example RNA Coverage Analysis Report:



ampliSeqRNA Plugin

The ampliSeqRNA Plugin generates statistics, downloadable data files, andinteractive visualization of targeted RNA amplicons.

Note: Use the ampliSeqRNA Plugin for expression analysis only. For gene fusionanalysis, use Ion Reporter™ Software.

There are two ways to run the ampliSeqRNA Plugin:• Automatically, by selecting and configuring the plugin during run planning or• Manually, allowing you to run the plugin at any time from a completed run

report.

1. To run the ampliSeqRNA plugin automatically during run analysis, select theampliSeqRNA checkbox in the Plugins screen in the template or Create PlanWizard. After selecting the checkbox, a Configure link appears:

2. Click Configure to open the ampliSeqRNA Plugin configuration dialog:

Configure and runthe ampliSeqRNAPlugin

Appendix F Data analysisampliSeqRNA Plugin F


3. Select the checkbox for one or both Filter Barcodes and ERCC Tracking.• Filter Barcodes − filters low frequency barcode reads, which might result

from barcode contamination, from subsequent analysis.• ERCC Tracking − is recommended if you spiked your RNA amplicon library

with ERCC tracking targets. The analysis produces an extra statistic forreads mapped to these targets so that the true level of "off-target" reads isaccurately represented. Your reference should include ERCC references forERCC reads to be included in the fraction of reads considered "on-target".

4. Click Save Changes.

Appendix F Data analysisampliSeqRNA PluginF


Safety

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.

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

G


Symbol English Français

On/Off On/Off (marche/arrêt)

Protective conductor terminal (mainground)

Borne de conducteur de protection(mise à la terre principale)

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 about responsibledisposal 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 paséliminer ce déchetélectronique avec les déchetsusuels non soumis au trisélectif. Se conformer auxordonnances locales sur lesdéchets municipaux pour lesdispositions d’élimination etcommuniquer avec le serviceà la clientèle pour desrenseignements sur lesoptions d’éliminationresponsable.

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 Low Voltage Directive2006/95/EC, EMC Directive 2014/30/EU, and R&TTE Directive1999/5/EC.

Indicates conformity with Australian standards for electromagneticcompatibility.

Conformitysymbols

Appendix G SafetySymbols on this instrumentG


Location of safety labels on this instrument

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.

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.

General

Appendix G SafetyLocation of safety labels on this instrument G


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.

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.

Physical injury

Electrical

Appendix G SafetyInstrument safetyG


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:

Reference Description

EU Directive2006/95/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 for measurement,control, and laboratory use – Part 1: General requirements

IEC 61010-2-010

EN 61010-2-010

Safety requirements for electrical equipment for measurement,control and laboratory use – Part 2-010: Particular requirementsfor laboratory equipment for the heating of materials

IEC/EN 61010-2-020 Safety requirements for electrical equipment for measurement,control and laboratory use – Part 2-020: Particular requirementsfor laboratory centrifuges

IEC 61010-2-081

EN 61010-2-081

Safety requirements for electrical equipment for measurement,control and laboratory use – Part 2-081: Particular requirementsfor automatic and semi-automatic laboratory equipment foranalysis and other purposes

Cleaning anddecontamination

Ultraviolet (UV)Safety

Safety compliance

Appendix G SafetySafety and electromagnetic compatibility (EMC) standards G



Directive 2014/30/EU 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

US FCC CFR Title 47Part 15.225, Subpart C

Operation within the band 13.110−14.010 MHz.

Industry Canada RSS210, Issue 8, Annex 2 EN302 291-1/2 V1.1.1

Licence-Exempt Radio Apparatus: Category I Equipment


Directive 2012/19/EU European Union “WEEE Directive” – Waste electrical andelectronic equipment

Directive 2011/65/EU European Union “RoHS Directive” – Restriction of hazardoussubstances in electrical and electronic equipment

EMC

Environmentaldesign

Appendix G SafetySafety and electromagnetic compatibility (EMC) standardsG


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. Consult the relevant SDSfor specific precautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by thechemical 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 G SafetyChemical safety G


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. Conduct all work in properly equipped facilitieswith the appropriate safety equipment (for example, physical containmentdevices). Safety equipment can also include items for personal protection, suchas gloves, coats, gowns, shoe covers, boots, respirators, face shields, safetyglasses, or goggles. Individuals should be trained according to applicableregulatory and company/ institution requirements before working withpotentially biohazardous materials. Follow all applicable local, state/provincial,and/or national regulations. The following references provide generalguidelines when handling biological samples in laboratory 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 G SafetyBiological hazard safetyG


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 thermofisher.com/support for the latest in services and support, including:• Worldwide contact telephone numbers• Product support, including:

– Product FAQs– Software, patches, and updates– Training for many applications and instruments

• 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.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact LifeTechnologies at www.thermofisher.com/support.


http://thermofisher.com/support

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/us/en/home/global/terms-and-conditions.html

http://www.thermofisher.com/support

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http://thermofisher.com/support

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