Ion AmpliSeq™ Library Preparation on the Ion

Chef™ System
USER GUIDE

for use with:

Ion AmpliSeq™ Chef-Ready Kits
Oncomine™ Chef-Ready Kits
Ion AmpliSeq™ On‑Demand Panels
Ion AmpliSeq™ Ready-to-Use Panels
Ion AmpliSeq™ Made-to-Order and Community Panels
Ion AmpliSeq™ Sample ID Panel
Ion Chef™ System

Catalog Number A29024

Publication Number MAN0013432
Revision H.0

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

 Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, California 92008 USA
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.

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

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

Revision history: Pub. No. MAN0013432

Revision Date Description
H.0 2 August 2021 • Corrected cycling conditions for the Ion AmpliSeq™ human and mouse
transcriptome panels.
• Support added for Ion AmpliSeq™ SARS‑CoV‑2 Insight Research Assay – GS Chef
Ready.

G.0 7 April 2021 • Updated Sample set requirements for Torrent Suite™ Software 5.16.
• Updated requirements for Oncomine™ Comprehensive Assay Plus.
• Support added for Oncomine™ Myeloid Research Assay – Chef‑Ready specific
workflow using Sample Sets.
• Support added for Ion AmpliSeq™ Transcriptome Mouse Gene Expression Panel,
Chef-Ready Kit.
• Information for Ion AmpliSeq™ Pharmacogenomics Research Panel, Chef-Ready
Kit removed due to discontinuation.
• Support added for Qubit™ 4 Fluorometer.
• Support added for Oncomine™ Tumor Specific Panels.
• Ion AmpliSeq™ On‑Demand panels updated.
• Support added for Ion 510™ and Ion 550™ Chips.
• Added instructions for removing deaminated bases from purified FFPE DNA.
• Instructions clarified for Made-to-Order Panels, spike-in panels, and Ion AmpliSeq
Sample ID Panels.
™
• Support added for the Ion AmpliSeq RNA ERCC Companion Panel.
• Content reorganization.

F.0 17 October 2017 • Product information table in “Ion AmpliSeq™ On‑Demand panels” updated with
new Ion AmpliSeq™ On‑Demand Panel configurations.
• Appendix F, “Expand any Ion AmpliSeq™ panel by adding a spike‑in panel” added.

E.0 16 May 2017 • Support added for Ion AmpliSeq™ On‑Demand Panels.
• Last two rows of the amplification cycle table merged, anneal/extension time
column added to table, and anneal/extension time recommendations modified for
higher plexy panels (see “Start the Ion Chef™ run” on page 31).

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these
products, you accept the 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
registered trademark of Roche Molecular Systems, Inc., used under permission and license. Eppendorf and LoBind are trademarks of
Eppendorf AG.
©2021 Thermo Fisher Scientific Inc. All rights reserved.
 Contents

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

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
™
Ion AmpliSeq Kit for Chef DL8 (library preparation) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
™ ™
Ion AmpliSeq and Oncomine Chef‑Ready Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
™
Ion AmpliSeq Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Recommended materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
™
Ion AmpliSeq Kit for Chef DL8 workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

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

Isolate DNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Guidelines for isolating and quantifying gDNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Guidelines for the amount of DNA needed per target amplification reaction . . . . . . . 16
Remove deaminated bases from purified FFPE DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Dilute the DNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
™
Add DNA to the IonCode PCR plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

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

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

Guidelines for isolating and quantifying RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Dilute the RNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Reverse transcribe RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ ™
CHAPTER 4 Run the Ion Chef System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Thaw the reagents and prepare the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Create a Sample Set—Imported file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Create a Sample Set manually . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
™
Set up the Ion Chef System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
™
Add Ion AmpliSeq 2X Primer Pools to Positions A and B of the
Reagents cartridge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
™
Load the Ion Chef Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
™
Start the Ion Chef run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

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

™
Unload the Ion Chef Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
™
(Optional) Combine libraries from multiple Ion Chef runs . . . . . . . . . . . . . . . . . . . . . . . 39
™
Clean the Ion Chef Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
About the cleaning protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
™
Clean the Ion Chef Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

■ CHAPTER 5 Templating, sequencing and data analysis . . . . . . . . . . . . . . . . . . . . . . 42

Create a Planned Run with Sample Sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

Templating, sequencing and data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

■ APPENDIX A Tips and troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

View troubleshooting and FAQs online . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
™
Ion Chef System setup and operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

■ ™
APPENDIX B Ion AmpliSeq panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
™
Oncomine tumor specific panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
™
Ion AmpliSeq On‑Demand Panel designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
™
Ion AmpliSeq Custom panel designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
™
Ion AmpliSeq Fixed Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

■ APPENDIX C Supplemental procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

™ ™
Reverse transcribe RNA using the SuperScript VILO cDNA Synthesis Kit . . . . . . . . . . . 52
™
(Optional) Qubit Fluorometer: Quantify the FFPE DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Create a samples file from a template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Sample attributes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

■ APPENDIX D Strategies for combining Ion AmpliSeq libraries . . . . . . . . . . . . . . 59

™

Combine barcoded libraries from multiple runs for sequencing on a single Ion chip . . . . . 59
Combine libraries prepared using different panels for equal coverage . . . . . . . . . . . . . . . . . 60
Combine unequal volumes of libraries for varying depth of coverage . . . . . . . . . . . . . . . . . 61
™
Ion Chip capacities for Ion AmpliSeq DNA libraries sequenced at equal depths . . . . . . . 62
™
Ion Chip capacities for Ion AmpliSeq RNA libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

■ ™
APPENDIX E Ion AmpliSeq Made-to-Order Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

Prepare primer pools from plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

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

■ APPENDIX F Expand any Ion AmpliSeq panel by adding a

™

spike‑in panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

■ ™
APPENDIX G Ion AmpliSeq Sample ID Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
™
Using the Ion AmpliSeq Sample ID Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

■ ™
APPENDIX H Ion AmpliSeq RNA ERCC Companion Panel . . . . . . . . . . . . . . . . . . 70

Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Software compatibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Contents and storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Required materials not supplied . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Add ERCC RNA Spike-In Mix 1 to RNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
™
Add the Ion AmpliSeq RNA ERCC Companion Panel to your RNA panel . . . . . . . . . . . . . 73
Supplemental procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Confirm ERCC_Analysis plugin version . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Install the ERCC_Analysis plugin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

■ APPENDIX I Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Symbols on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Conformity symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Location of safety labels on this instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Safety information for instruments not manufactured by Thermo Fisher Scientific . . . . . . 77
Instrument safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Physical injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Electrical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Cleaning and decontamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Ultraviolet (UV) Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Safety and electromagnetic compatibility (EMC) standards . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Safety compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
EMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Environmental design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 5
 1 Product information

CAUTION! ABBREVIATED SAFETY ALERTS. Hazard symbols and hazard types specified in
procedures may be abbreviated in this document. For the complete 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) is used for automated library
preparation of Ion AmpliSeq™ libraries using the Ion Chef™ System. The kit provides sufficient material
for the preparation of 32 libraries (4 Ion Chef™ runs).
Key features of the kit include:
• Automated workflow for library preparation of up to 8 barcoded Ion AmpliSeq™ libraries per
Ion Chef™ run using a single 1- or 2-pool Ion AmpliSeq™ panel.
• Easy-to-use cartridge format with reagent barcoding for full reagent tracking.
• Proven performance of Ion AmpliSeq™ technology, compatible with DNA extracted from formalin-
fixed, paraffin-embedded tissue and other challenging sources.

Software compatibility
The Ion AmpliSeq™ Kit for Chef DL8 is compatible with Torrent Suite™ Software 5.0 and later. We
recommend updating your Torrent Server, Ion Chef™, and Ion sequencer instrument software to the
latest available versions before using this kit.
If you are using the Oncomine™ Comprehensive Assay Plus or the Oncomine™ Myeloid Research Assay,
see the user guide for your assay for software compatibility requirements.

6 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 1 Product information
Ion AmpliSeq™ Kit for Chef DL8 (library preparation) 1

Ion AmpliSeq™ Kit for Chef DL8 (library preparation)

IMPORTANT! Store all consumables and cartridges under the recommended conditions and in an
upright position. Ion AmpliSeq™ Chef Solutions DL8 cartridges are 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 required for performing
4 Ion Chef™ runs, with up to 8 Ion AmpliSeq™ libraries prepared per run. Upon arrival, inspect all
consumables and contact Technical Support if any of the components have been damaged during
shipping.

Quantity per
Component Part No. Storage
kit

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) A29027 1 box with 15°C to 30°C
™
• Ion AmpliSeq Tip Cartridge L8 4 inserts

• PCR Frame Seal

• Enrichment Cartridge

IonCode™ 0101–0132 in 96 Well PCR Plates (dried) A29028 1 set of 15°C to 30°C
4 plates
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)

[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.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 7
 Chapter 1 Product information
1 Ion AmpliSeq™ Kit for Chef DL8 (library preparation)

6 7

8 9

™
Ion AmpliSeq Kit for Chef DL8 consumables

1 Ion AmpliSeq™ Chef Reagents DL8 cartridge

2 Ion AmpliSeq™ Chef Solutions DL8 cartridge
3 Ion AmpliSeq™ Tip Cartridge L8
4 Enrichment Cartridge
5 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.

8 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 1 Product information
Ion AmpliSeq™ and Oncomine™ Chef‑Ready Kits 1

Ion AmpliSeq™ and Oncomine™ Chef‑Ready Kits

• Ion AmpliSeq™ and Oncomine™ Chef-Ready Kits contain assays packaged with the reagents and
supplies from the stand-alone Ion AmpliSeq™ Kit for Chef DL8 (see “Ion AmpliSeq™ Kit for Chef
DL8 (library preparation)” on page 7 for details).
• Oncomine™ tumor specific panels (AmpliSeq.com) can be used with the Ion AmpliSeq™ Kit for
Chef DL8.
• Each Chef‑Ready Kit contains sufficient reagents to prepare 32 libraries (4 runs) on the Ion Chef™
System.
• For assays that include both DNA and RNA panels, DNA and RNA libraries must be prepared on
separate runs of the Ion Chef™ System, each with a different set of IonCode™ Barcode Adapters.
• Store Ion AmpliSeq™ and Oncomine™ panels at –30°C to –10°C . Store the Ion AmpliSeq™ Kit for
Chef DL8 as directed (see “Ion AmpliSeq™ Kit for Chef DL8 (library preparation)” on page 7).

Assays Primer pools[1] Panel type Quantity

Ion AmpliSeq™ Chef‑Ready Kits

Ion AmpliSeq™ SARS‑CoV‑2 Insight Research Assay – GS Chef 2 RNA 8 tubes

Ready (Cat. No. A51306)

Ion AmpliSeq™ Cancer Hotspot Research Panel v2, Chef-Ready 1 DNA[2]

Kit (Cat. No. A32914)

Ion AmpliSeq™ Transcriptome Human Gene Expression Panel, 1 RNA[3]

Chef-Ready Kit (Cat. No. A31446)

Ion AmpliSeq™ Transcriptome Mouse Gene Expression Panel, 1 RNA[4]

Chef-Ready Kit (Cat. No. A36412)

Oncomine™ Chef‑Ready Kits

Oncomine™ BRCA Research Assay Chef-Ready Library 2 DNA 8 tubes

Preparation (Cat. No. A32841)

Oncomine™ Tumor Mutation Load Assay – Chef‑Ready Library 2 DNA

Preparation (Cat. No. A37910)

Oncomine™ Immune Response Research Assay 1 RNA

(Cat. No. A32928 )

Oncomine™ Myeloid Research Assay – Chef‑Ready Library 2 (DNA) DNA and RNA
Preparation (Cat. No. A36941) 1 (RNA)

Oncomine™ Comprehensive Assay v3C (Cat. No. A35806 ) 2 (DNA) DNA and RNA
2 (RNA)

Oncomine™ Comprehensive Assay Plus – Automated Library 2 (DNA) DNA and RNA
Preparation (Cat. No. A49667) 2 (RNA)

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 9
 Chapter 1 Product information
1 Required materials not supplied

(continued)

Assays Primer pools[1] Panel type Quantity

Oncomine™ Childhood Cancer Research Assay–Chef-Ready 2 (DNA) DNA and RNA 8 tubes
(Cat. No. A36486) 2 (RNA)

Oncomine™ Focus Assay, Chef‑Ready (Cat. No. A42008) 1 (DNA) DNA and RNA
1 (RNA)
[1] Each primer pool is supplied at the appropriate volume and concentration for directly loading into an Ion AmpliSeq™ Kit for Chef DL8 cartridge.
[2] 207 primer pairs per pool
[3] 20,800 primer pairs per pool
[4] 23,930 primer pairs per pool

Required materials not supplied

Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.

Item Source

Ion Chef™ System 4484177

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

One of the following:

• SuperScript™ IV VILO™ Master Mix • 11756050
™ ™
• SuperScript IV VILO Master Mix with ezDNase Enzyme ™
• 11766050
• Ion Torrent™ NGS Reverse Transcription Kit[2] • A45003
• SuperScript™ VILO™ cDNA Synthesis Kit[3] • 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.
[2] Recommended for the Oncomine™ Comprehensive Assay Plus – Automated Library Preparation and the Oncomine™ tumor specific
RNA panel.
[3] Recommended for the Ion AmpliSeq™ Transcriptome Human Gene Expression Panel, Chef-Ready Kit, Ion AmpliSeq™
Transcriptome Mouse Gene Expression Panel, Chef-Ready Kit, and the Oncomine™ Focus Assay, Chef‑Ready Kit.

10 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 1 Product information
Required materials not supplied 1

Ion AmpliSeq™ Panels

Ion AmpliSeq™ panels provide pools of primers for the amplification of target regions. The primers
contain proprietary modifications that enable removal of primer sequences during library preparation for
efficient target assessment during sequencing. Multiple primer pools can be used to create overlapping
amplicons that enable complete coverage of large targets. Panels are designed to be used with nucleic
acid from various sources, including nucleic acid from formalin‐fixed paraffin‐embedded (FFPE) tissue
and cell-free DNA (cfDNA).
Ion AmpliSeq™ panels are ordered using Ion AmpliSeq™ Designer. Users who are interested in inherited
disease or germline research applications can design and order panels using a content selection engine
or by uploading their own gene list. The gene designs in the On-Demand catalog have been optimized
for high performance. For additional information, see Appendix B, “Ion AmpliSeq™ panels” or visit
AmpliSeq.com.
The following types of panels are compatible with the Ion Chef™ System.

In stock Made–to–order Fixed panels

Product and Oncomine™ Ion AmpliSeq™ Ion AmpliSeq™ Ion AmpliSeq™
description Ion AmpliSeq™
tumor specific On‑Demand Community Ready-to-Use
Custom panels
panels Panels Panels Panels

Genome Human Human Any Human Human

Nucleic Acid DNA/RNA DNA only DNA/RNA DNA/RNA DNA/RNA

Sample type FFPE Genomic DNA Any Any Any

Target type Genes Genes Genes, regions, Genes, regions, Genes, regions,
hotspots, hotspots, hotspots,
amplicons, amplicons, amplicons,
expression, expression, expression,
fusions fusions fusions

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 11
 Chapter 1 Product information
1 Recommended materials and equipment

Recommended materials and equipment

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

Item Source

Additional equipment

Fisher Scientific™ Mini Plate Spinner Centrifuge, 14‑100‑143

or equivalent 96-well plate centrifuge (fisherscientific.com)

Recommended for nucleic acid isolation

Ion AmpliSeq™ Direct FFPE DNA Kit A31133

RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE AM1975

RecoverAll™ Multi-Sample RNA/DNA Workflow A26069 and A26135

MagMAX™ FFPE DNA/RNA Ultra Kit A31881

PureLink™ Genomic DNA Mini Kit K182001

PureLink™ RNA Mini Kit 12183018A

Recommended for nucleic acid quantification

TaqMan™ RNase P Detection Reagents Kit (Recommended for gDNA) 4316831

Qubit™ 4 Fluorometer[1] and one or more of the following kits: Q33238

™
• Qubit dsDNA HS Assay Kit (DNA) • Q32851, Q32854
™
• Qubit RNA HS Assay Kit (RNA) • Q32852, Q32855

(Optional) Ion Library TaqMan™ Quantitation Kit[2] 4468802

Additional material for the removal of deaminated bases from purified FFPE DNA

Uracil DNA Glycosylase (UDG), heat labile 78310100UN

[1] Qubit™ 2.0 Fluorometer and later are supported.
[2] Recommended for checking concentration of combined library.

12 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 1 Product information
Ion AmpliSeq™ Kit for Chef DL8 workflow 1

Ion AmpliSeq™ Kit for Chef DL8 workflow

The following workflow summarizes the preparation of DNA and RNA Ion AmpliSeq™ libraries using the
Ion AmpliSeq™ Kit for Chef DL8 and the Ion Chef™ System.

1 Prepare DNA Samples

Remove deaminated bases from purified FFPE

DNA (page 16)
FFPE preservation methods can lead to significant cytosine deamination
within the isolated DNA, and result in decreased sequencing quality.
As a result, we recommend removing deaminated cytosines before
sequencing.

Dilute the DNA samples (page 17)

DNA samples are diluted to an optimal concentration.

Add DNA to the IonCode™ PCR plate (page 18)

DNA is added to the barcode plate for library preparation.
When complete, proceed to “Run samples” 3 .

2 Prepare RNA Samples

Isolate and dilute RNA samples (page 19)

Guidelines for isolated RNA are described. RNA is diluted to an optimal
concentration.

Reverse transcribe RNA (page 20)

RNA is added to the barcode plate and reverse transcribed to generate
cDNA for library preparation.
When complete, proceed to “Run samples” 3 .

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 13
 Chapter 1 Product information
1 Ion AmpliSeq™ Kit for Chef DL8 workflow

3 Run samples

Create a sample set (page 24)

You can use the Sample Set to create a Planned Run. The information
from the Sample Set and individual samples within the Sample Set are
prepopulated in the Planned Run workflow bar steps and Planned Run
template.

Add Ion AmpliSeq™ 2X Primer Pools to Positions A

and B of the Reagents cartridge (page 26)
Add Ion AmpliSeq™ Panel to the gDNA or cDNA samples for library
preparation.

Load the Ion Chef™ Instrument (page 28)

Install reagents and consumables on the Ion Chef™ Instrument.

Start the Ion Chef™ run (page 31)

Enter panel specific amplification parameters, perform automated library
preparation and pooling of barcoded sample libraries on the Ion Chef™
Instrument.

Unload the Ion Chef™ Instrument (page 38)

Unload completed library pools for sequencing and clean the Ion Chef™
Instrument.

14 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 2 Prepare DNA samples

■ Isolate DNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■ Remove deaminated bases from purified FFPE DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
■ Dilute the DNA samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
■ Add DNA to the IonCode™ PCR plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

We recommend that you create a sample set, 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 the
instrument immediately after transfer to the IonCode™ 96‑well PCR plate (see Chapter 4, “Run the
Ion Chef™ System”).
For information on preparing RNA samples, go to Chapter 3, “Prepare RNA samples”.

IMPORTANT! If you are using an Oncomine™ Chef-Ready Kit, follow the DNA sample preparation
procedure in the user guide for your assay.

Isolate DNA samples

Guidelines for isolating and quantifying gDNA
• High-quality gDNA can be prepared and quantified using commercially available kits. See
“Required materials not supplied” on page 10 for kits that are recommended 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 used for quantification,
particularly for FFPE DNA, and highly degraded DNA samples.
• Methods such as densitometry (for example, NanoDrop™ spectrophotometers) are not
recommended, because they are not specific for gDNA, and are therefore sensitive to small
fragments of hydrolyzed RNA. Using densitometry can lead to overestimation of the sample gDNA
concentration, under-seeding of the target amplification reaction, low library yields, and poor chip
loading.
• The Direct FFPE DNA preparation can be stored for up to 6 months at –20°C before library
preparation.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 15
 Chapter 2 Prepare DNA samples
2 Remove deaminated bases from purified FFPE DNA

Guidelines for the amount of DNA needed per target amplification reaction
• Select one of the following guidelines for input DNA.
– Oncomine™ kits only: for each target amplification reaction, use 300–30,000 copies of DNA
(10 ng of mammalian gDNA) from normal or FFPE tissue.
– All other kits: for each target amplification reaction, use 300–30,000 copies of DNA (1−100 ng
of mammalian gDNA) from normal tissue, FFPE tissue, or cfDNA.
• Increasing the amount of DNA results in higher-quality libraries, especially when DNA quality or
quantity is unknown. We recommend using 1 ng gDNA (300 copies) only with high-quality, well-
quantified samples.

Remove deaminated bases from purified FFPE DNA

IMPORTANT! This procedure is required for the Oncomine™ Comprehensive Assay Plus and
Oncomine™ Tumor Mutation Load Assay. Follow the instructions in the user guide for your assay.

If using DNA isolated from sources other than FFPE tissue, proceed directly to “Dilute the DNA
samples” on page 17.
FFPE preservation methods can lead to significant cytosine deamination of the isolated DNA, resulting
in decreased sequencing quality. Deaminated cytosine (uracil) bases can be enzymatically removed by
treatment with Uracil DNA Glycosylase (UDG) before the target amplification reaction.

Note: We recommend treating all samples including FFPE and high quality DNA (for example,
commercial controls or DNA isolated from cell lines) with UDG to remove deaminated bases before
target amplification.

1. For each FFPE DNA sample, add the following components to a single well of a 96‑well PCR plate.

Component Volume
20 ng FFPE DNA ≤9 µL
UDG, heat‑labile 1 µL
Low TE to 10 µL

2. Mix the reaction by pipetting at least half the total volume up and down at least 5 times, then seal
the plate with MicroAmp™ Clear Adhesive Film. Alternatively, seal the plate, vortex for 5 seconds to
mix the reactions, then centrifuge briefly to collect the contents.

Note: To prevent evaporation during UDG treatment, use an applicator tool to press the film
securely around each reaction well and around the perimeter of the plate.

16 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 2 Prepare DNA samples
Dilute the DNA samples 2

3. Place a MicroAmp™ Optical Film Compression Pad on the plate, load the plate into the thermal
cycler, then run the following program.

Temperature Time
37°C 2 minutes
50°C 10 minutes
4°C Hold (≤1 hour)

4. Remove the plate from the thermal cycler, then centrifuge briefly to collect the contents.

STOPPING POINT Reactions can be stored at −20°C long term.

5. Carefully remove the plate seal, add 20 μL of nuclease–free water to each well, mix the reaction by
pipetting at least half the total volume up and down at least 5 times, then proceed immediately to
“Add DNA to the IonCode™ PCR plate” on page 18. Add 15 µL of the UDG treated DNA to the
target amplification reaction (for 10 ng input).

Dilute the DNA samples

Dilute samples to 0.67 ng/µL with Nuclease-free Water. Prepare 15 µL of each diluted sample (10 ng) to
make an Ion AmpliSeq™ Chef library.

Note:
™
· If you are using an Oncomine Chef–Ready Kit, see the user guide for your assay for instructions for
diluting the DNA samples.
· 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.
· you choose to use less gDNA, decrease the concentration, not the volume. For example, to apply
If
1 ng of gDNA per library, use 15 µL of gDNA at 0.067 ng/µL.
· If you are using Direct FFPE DNA, do not dilute the lysate.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 17
 Chapter 2 Prepare DNA samples
2 Add DNA to the IonCode™ PCR plate

Add DNA to the IonCode™ PCR plate

1. Remove, then 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 DNA sample, into wells A1 to
H1 of the plate.

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

E
F
G

1 2

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 barcode number is in A6, and the
highest barcode number is in H6. All appear light blue in the actual plates.

Note:
· If you are processing fewer than 8 samples, it is preferable to add replicates or positive control
samples to the run. Otherwise, pipet 15 µL of Nuclease-free Water as non-template control into
column 1 wells that do not contain a DNA sample.
· If processing 5 or fewer samples, we recommend that you quantify the output combined library
by qPCR to ensure that an optimal concentration is used in templating reactions.

3. Carefully inspect each well for air bubbles. Remove any air bubbles by gentle pipetting.
Alternatively, seal the plate with MicroAmp™ Clear Adhesive Film, then centrifuge the plate briefly in
a plate centrifuge. Carefully remove the plate seal.
IMPORTANT!
· If you are sealing the plate, offset the film to the left so that the adhesive does not cover the
barcode label. If the barcode label becomes damaged, you can override the error during the
Deck Scan.
™ ™
· If using the Ion AmpliSeq Direct FFPE DNA Kit, start the Ion Chef run within 10 minutes of
™
transferring the last sample slurry to the IonCode 96‑well PCR plate.
· ≥10 minutes has elapsed, pipet each sample slurry up and down at least 5 times to mix, load
If
the IonCode™ 96‑well PCR plate onto the Ion Chef™ Instrument, then start the run.

Proceed to Chapter 4, “Run the Ion Chef™ System”.

18 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 3 Prepare RNA samples

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

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

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

IMPORTANT! If you are using an Oncomine™ Chef-Ready Kit or the Ion AmpliSeq™ SARS‑CoV‑2
Insight Research Assay – GS Chef Ready, follow the RNA sample preparation procedure in the user
guide for your assay.

Isolate and dilute RNA samples

Guidelines for isolating and quantifying RNA
• High-quality RNA can be prepared and quantified using commercially available kits. See “Required
materials not supplied” on page 10 for kits recommended for isolating RNA from fresh and FFPE
samples.
• We recommend the Qubit™ RNA HS Assay Kit (Cat. No. Q32855) for quantifying RNA.
• Methods such as densitometry (for example, NanoDrop™ spectrophotometers) are not
recommended, because they are sensitive to small fragments of hydrolyzed RNA. Using
densitometry can lead to overestimation of the sample RNA concentration, under-seeding of the
target amplification reaction, low library yields, and poor chip loading.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 19
 Chapter 3 Prepare RNA samples
3 Reverse transcribe RNA

Dilute the RNA samples

Dilute samples with Nuclease-free Water according to the following table, depending on the reverse
transcription enzyme you are using. You must prepare either 12 µL, 7 μL, or 10.5 µL of each diluted
sample (10 ng) for reverse transcription reactions before an Ion AmpliSeq™ Chef library preparation run.

Volume of RNA required

Reverse transcriptase RNA concentration
per reaction
SuperScript™ IV VILO™ Master Mix 0.83 ng/µL 12 µL
Ion Torrent™ NGS Reverse Transcription Kit 1.43 ng/µL 7 µL
SuperScript VILO cDNA Synthesis Kit[1]
™ ™
0.95 ng/µL 10.5 µL
[1] Recommended for the Human and Mouse Ion AmpliSeq™ Transcriptome panels (Cat. Nos. A31446 and A36412).

Note: If you choose to use more RNA, increase the concentration, not the volume. For example, for
the SuperScript™ VILO™ Master Mix reaction, to apply 100 ng of RNA per library, use 12 µL of RNA at
8.33 ng/µL, or 10.5 µL of RNA at 9.5 ng/µL.

Proceed to reverse transcription (“Reverse transcribe RNA” or “Reverse transcribe RNA using the
SuperScript™ VILO™ cDNA Synthesis Kit” on page 52).

Reverse transcribe RNA

RNA samples must be manually converted to cDNA before library preparation on the Ion Chef™
Instrument. We recommend using the Ion Torrent™ NGS Reverse Transcription Kit or SuperScript™
IV VILO™ Master Mix (ordered separately), which simplifies reaction setup and provides improved
performance. If you are using an Oncomine™ Chef–Ready Kit, see the user guide for your assay to
determine the correct reverse transcriptase.

Note:
™
· For setting up reverse transcription reactions with the SuperScript III Reverse Transcriptase, see
“Reverse transcribe RNA using the SuperScript™ VILO™ cDNA Synthesis Kit” on page 52.
· Set up the reverse transcription reactions on ice or a cold block.

1. If you are using the ERCC RNA Spike‑In Mix and the Ion AmpliSeq™ RNA ERCC Companion Panel
proceed to “Add ERCC RNA Spike-In Mix 1 to RNA samples” on page 72.

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

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

4. Add reverse transcription mix.

• Pipet 3 µL of the SuperScript™ IV VILO™ Master Mix into wells A1 to H1 of the plate.
• Pipet 2 µL of the NGS 5X Reaction Buffer into wells A1 to H1 of the plate, then pipet 1 µL of
the NGS 10X RT Enzyme Mix into wells A1 to H1 of the plate.

20 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 3 Prepare RNA samples
Reverse transcribe RNA 3

5. Add RNA.
• If you are using the Ion Torrent™ NGS Reverse Transcription Kit, pipet 7 µL of each RNA
sample (1.43 ng/µL, 10 ng total) into column 1 wells containing the reverse transcription mix to
give a 10‑µL reaction volume.
• If you are using the SuperScript™ VILO™ Master Mix, pipet 12 µL of each RNA sample
(0.83 ng/µL, 10 ng total) into column 1 wells containing the RT Master Mix to give a 15‑µL
reaction volume.

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

E
F
G

1 2

1 Each column 1 well contains 3 µL SuperScript™ IV VILO™ Master Mix or , and 12 µL of diluted 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 barcode number 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 or positive control
samples to the run. Otherwise, pipet 12 µL of Nuclease-free Water as non-template control into
column 1 wells that do not contain an RNA sample.

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

7. Seal the plate with a MicroAmp™ Clear Adhesive Film, vortex thoroughly, then briefly centrifuge the
plate to collect droplets. Alternatively, mix by pipetting at least 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 the barcode label. If
the barcode label becomes damaged, you can override the error during Deck Scan.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 21
 Chapter 3 Prepare RNA samples
3 Reverse transcribe RNA

8. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal cycler, 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 the thermal 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 any condensate.
Alternatively, centrifuge briefly to collect droplets.

Proceed to “Thaw the reagents and prepare the instrument” on page 23.

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

■ Thaw the reagents and prepare the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

■ Create a Sample Set—Imported file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
■ Create a Sample Set manually . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
■ Set up the Ion Chef™ System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
■ Start the Ion Chef™ run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
■ Unload the Ion Chef™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
■ Clean the Ion Chef™ Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

IMPORTANT! If you are using the Ion AmpliSeq™ SARS‑CoV‑2 Insight Research Assay – GS Chef
Ready, follow the recommendation in the Ion AmpliSeq™ SARS‑CoV‑2 Insight Research Assay User
Guide (Pub. No. MAN0024915).

Thaw the reagents and prepare the instrument

• Before the run, thaw one Ion AmpliSeq™ Chef Reagents DL8 cartridge at room temperature 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 40 ).

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

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

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 23
 Chapter 4 Run the Ion Chef™ System
4 Create a Sample Set—Imported file

Create a Sample Set—Imported file

IMPORTANT!
™ ™
· If you are using the Oncomine Comprehensive Assay Plus or the Oncomine Myeloid Research
Assay – Chef‑Ready, follow the recommendations for creating sample sets in the user guide for your
assay.
™
· If you are using Torrent Suite Software version 5.16 or later, you must create a sample set.

You can use the Sample Set to create a Planned Run. The information from the Sample Set and
individual samples within the Sample Set are prepopulated in the Planned Run workflow bar steps and
Planned Run template. For more information, see the help system for your version of Torrent Suite™
Software.
To create an Ion AmpliSeq™ on Ion Chef™ Sample Set, you can either import samples from a CSV file, or
enter them manually.
To download a template CSV file, see “Create a samples file from a template” on page 54.
To create a sample set manually, see “Create a Sample Set manually” on page 25.

Note: For more information, see the help system for your version of Torrent Suite™ Software.

1. In the Plan tab, click Samples, then click Import Samples from File.

2. In the Import Samples screen, click Sample File Format.

A CSV template file downloads to your computer in the Downloads folder.

3. Open the CSV template file, then enter the sample names, PCR plate positions, and barcodes
used. Save the file to your computer with a new name.
You can also enter sample names in the CSV file, then supply the plate position, barcode, and
other information later by editing the Sample Set in the Sample Sets screen.

4. Click Select File, select your new CSV file, then click Open.

5. Click Add Sample Set.

6. Complete the Add Sample Set dialog box.

a. Enter a Sample Set name.

24 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 4 Run the Ion Chef™ System
Create a Sample Set manually 4

b. Select the appropriate Group Type.

c. Set Library Prep Type to AmpliSeq on Chef.

d. Set Library Prep Kit to Ion AmpliSeq Kit on Chef

DL8.

e. Type or scan the PCR plate serial number.

f. In Description, enter additional information.

g. Click Save & Finish

A new Sample Set is created.

Proceed to “Set up the Ion Chef™ System” on page 26.

Create a Sample Set manually

IMPORTANT!
™ ™
· If you are using the Oncomine Comprehensive Assay Plus or the Oncomine Myeloid Research
Assay – Chef‑Ready, follow the recommendations for creating sample sets in the user guide for your
assay.
™
· If you are using Torrent Suite Software version 5.16 or later, you must create a sample set.

You can use the Sample Set to create a Planned Run. The information from the Sample Set and
individual samples within the Sample Set are prepopulated in the Planned Run workflow bar steps and
Planned Run template. For more information, see the help system for your version of Torrent Suite™
Software.
Create a Sample Set manually by entering sample information into the Torrent Suite™ Software without
the use of an external CSV file. This method of entering sample information is useful for creating small
Samples Sets.
To create a Sample Set manually, enter individual samples into the software, then create a new Sample
Set and add your samples to it. Alternatively, you can add your new samples to an existing Sample Set.
For Sample Sets that contain numerous samples, you may want to import samples using a CSV file. For
more information, see “Create a Sample Set—Imported file” on page 24.

1. In the Plan tab, click Samples, then click Add or Update Sample Set/Samples.

2. Click Enter New Sample.

a. Complete the Add Sample dialog box. For information about defining the samples, see the
help system for your version of Torrent Suite™ Software.

b. Click Done.
Your new samples and sample attributes appear in the Enter Samples list.

c. Enter additional samples if needed.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 25
 Chapter 4 Run the Ion Chef™ System
4 Set up the Ion Chef™ System

3. Add the samples to a sample set. Do one of the following:

• Click Add to existing Sample Set, then select an existing Sample Set to contain the samples.
• Click Create Sample Set. For information about defining the Sample Set, see the help system
for your version of Torrent Suite™ Software.

4. Click Save Sample Set.

The set name appears in the Sample Sets list.

Proceed to “Set up the Ion Chef™ System” on page 26.

Set up the Ion Chef™ System

Note: For assays, with DNA and RNA panels, DNA and RNA libraries must be prepared on separate
runs of the Ion Chef™ System, each with a different set of IonCode™ Barcode Adapters.

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

cartridge
1. Uncap all 4 tubes in positions A, B, C, and D in the Ion AmpliSeq™ Chef Reagents DL8 cartridge.
Save the caps.
• Standard panels

A 1

B 2

C 3

D 4

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)

26 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 4 Run the Ion Chef™ System
Set up the Ion Chef™ System 4

• Oncomine™ Comprehensive Assay Plus, DNA and RNA panels

A 1

B 2

C 3

D 4

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 (Output tube)
4 Position D (Output tube)

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

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

If you are using Action

Chef-Ready panels[1]
1 or 2 primer 1. Vortex the Primer Pool Tubes to mix, then centrifuge.
pools
2. Remove the caps, then replace the tubes in Positions A and B of the Ion
AmpliSeq™ Chef Reagents DL8 cartridge with the Chef-Ready panel tubes.

Ion AmpliSeq™ 2X panels—Aliquot only.

1 primer pool 1. Pipet 150 µL of the 2X Primer Pool into each of the Position A and Position B
tubes.

2 primer pools 1. Pipet 150 µL of the 2X Primer Pool 1 into the Position A tube.
2. Pipet 150 µL of the 2X Primer Pool 2 into the Position B tube.

Ion AmpliSeq™ 5X panels—Dilute and Aliquot.

1 primer pool 1. Dilute to 2X by adding 120 µL of the 5X Primer Pool to 180 µL of Nuclease-free
Water.
2. Pipet 150 µL of the 2X Primer Pool into each of the Position A and B tubes.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 27
 Chapter 4 Run the Ion Chef™ System
4 Set up the Ion Chef™ System

(continued)
If you are using Action
2 primer pools 1. Pipet 60 µL of the 5X Primer Pool 1 into the Position A tube.
2. Pipet 60 µL of the 5X Primer Pool 2 into the Position B tube.
3. Pipet 90 µL of Nuclease-free Water into each of the Position A and Position B
tubes.
4. Using a new tip for each tube, pipet up and down 5 times to mix.

[1] Not all Chef-Ready panels are provided at 2X.

Note:
· For standard panels, the tube in Position D contains 700 µL of combined barcoded libraries at
completion of the run.
™
· For the Oncomine Comprehensive Assay Plus, the tubes in Position C (samples A1–A4) and
Position D (samples A5–A8) contain 350 μL of combined barcoded libraries at the completion of
the run.

Load the Ion Chef™ Instrument

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

3 4 6

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

5
D

2 H

1 Ion AmpliSeq™ Chef Solutions DL8 cartridge 5 IonCode™ 96 Well PCR Plate
2 Ion AmpliSeq™ Chef Reagents DL8 cartridge 6 Empty Tip Cartridge L8
3 Ion AmpliSeq™ Tip Cartridge L8 7 Enrichment Cartridge
4 PCR Frame Seal

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

28 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 4 Run the Ion Chef™ System
Set up the Ion Chef™ System 4

1. Open the Ion Chef™ Instrument 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.

IMPORTANT! Lift the door from the center.

1 Center of door

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 is level 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 on the side of the tubes.

b. Load the cartridge into the Reagents station so that it snaps into place and is level on the
deck.
IMPORTANT! Ensure that 4 flagged tubes are uncapped and loaded in Positions A–D of the
Reagents cartridge, and 2X Primer Pools are loaded in Positions A and B.

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

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 to expose the pipette tips.

b. Slide the catch forward to allow the locking bracket to pivot upward. Load the Ion AmpliSeq™
Tip Cartridge L8 into position, pull the bracket downward, then push the catch backward to
lock the cartridge in place.
IMPORTANT! The run fails if you do not close the locking bracket.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 29
 Chapter 4 Run the Ion Chef™ System
4 Set up the Ion Chef™ System

6. If you are preparing libraries from RNA, carefully remove the plate seal from the IonCode™ 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, then carefully peel the seal with one
hand while firmly holding the plate and rack down on the bench with your other hand. Inspect
the column 1 wells for droplets on the side of wells. Apply a new plate seal and recentrifuge the
plate to collect droplets to the bottom of the wells, if needed.

7. (RNA samples only) If the samples were reverse transcribed using the Ion Torrent™ NGS Reverse
Transcription Kit, raise the volume to 15 μL.
a. Add 5 μL of nuclease-free water to each sample.

b. Seal the plate with a MicroAmp™ Clear Adhesive Film, vortex thoroughly, then briefly
centrifuge the plate to collect droplets. Alternatively, mix by pipetting at least 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 the barcode
label. If the barcode label becomes damaged, you can override the error during Deck Scan.

c. Carefully remove the plate seal.

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

9. Slide a new PCR Frame Seal underneath the automated heated cover.
IMPORTANT! When the PCR Frame Seal is positioned correctly, its tabs project upward and
contact the heated cover.

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

11. Close the instrument door by first lifting it up slightly to disengage the locking mechanism, then
pushing down on the door so that the lower locks engage.
IMPORTANT! After closing the door, ensure that both sides of the door are locked down.

30 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 4 Run the Ion Chef™ System
Start the Ion Chef™ run 4

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 deck loading steps on the instrument touch
screen.

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

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 31
 Chapter 4 Run the Ion Chef™ System
4 Start the Ion Chef™ run

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

6. On the Data Destination screen, ensure that the Server and Sample set information is correct, then
touch Next.

Note: If the PCR plate is not recognized, select the appropriate plate when prompted. If no sample
set was selected or planned in the Torrent Server, the following warning appears: "No sample Set
detected. Do you want to continue?" You must create and select a sample set. (See “Create a
Sample Set—Imported file” on page 24 or “Create a Sample Set manually” on page 25.)

• Example of a generic assay setup.

• Example of a correct Oncomine™ Comprehensive Assay Plus setup—When starting the library
preparation run on the Ion Chef™ Instrument ensure that the correct Kit Type and Sample set
are selected, and that 2 Library Pools - OCA Plus is displayed in order to properly prepare
Oncomine™ Comprehensive Assay Plus–Chef Ready libraries. If 2 Library Pools - OCA Plus
is not displayed the default library preparation script is run which results in all 8 libraries
combined into a single pool. The 2 Library Pools - OCA Plus library preparation script is only
available in Torrent Suite™ Software 5.16 or later.

Ensure that 2 Library Pools - OCA Plus appears below the Sample set dropdown list.

32 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 4 Run the Ion Chef™ System
Start the Ion Chef™ run 4

• Example of a correct Oncomine™ Myeloid Assay setup—When starting the library preparation
run on the Ion Chef™ Instrument ensure that the correct Kit Type and Sample set are
selected, and that Myeloid is displayed in order to properly prepare Oncomine™ Myeloid
Assay–Chef Ready libraries. If Myeloid is not displayed the default library preparation script is
run which may result in low quality libraries. The Myeloid specific library preparation script is
only available in Torrent Suite™ Software 5.14 or later.

Ensure that Myeloid appears below the Sample set dropdown list.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 33
 Chapter 4 Run the Ion Chef™ System
4 Start the Ion Chef™ run

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

Recommended number of amplification cycles for Ion AmpliSeq™ panels

Recommended number of amplification cycles

Primer pairs per pool (10 ng DNA/RNA, 3,000 copies)[2]
Anneal/Extension
(excluding gene fusion Low quality DNA/RNA time[3]
primer pairs)[1] High quality DNA/RNA (FFPE DNA/RNA or
cfDNA/RNA)
Panels for gene fusion 28 31 4 minutes
detection only
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

34 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 4 Run the Ion Chef™ System
Start the Ion Chef™ run 4

Recommended number of amplification cycles for Ion AmpliSeq panels (continued)

Recommended number of amplification cycles
Primer pairs per pool (10 ng DNA/RNA, 3,000 copies)[2]
Anneal/Extension
(excluding gene fusion Low quality DNA/RNA time[3]
primer pairs)[1] High quality DNA/RNA (FFPE DNA/RNA or
cfDNA/RNA)
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.
[3] For Ion AmpliSeq™ panels using a 375-bp amplicon design, use 8 minutes.

Recommended number of amplification cycles for Ion AmpliSeq™ and Oncomine™ Research
Assay Chef-Ready panels

Recommended number of
amplification cycles Anneal/
Assay (10 ng DNA/RNA, 3,000 copies) extension time
High quality Low quality (minutes)
DNA/RNA DNA/RNA[1]
Ion AmpliSeq™ Transcriptome Human 13 17 16
Gene Expression Panel, Chef-Ready Kit
(Cat. No. A31446)
Ion AmpliSeq™ Transcriptome Mouse 13 17 16
Gene Expression Panel, Chef-Ready Kit
(Cat. No. A36412)
Oncomine™ Tumor Mutation Load 15 16 16
Assay – Chef‑Ready Library Preparation
Kit (Cat. No. A37910)
Oncomine™ Immune Response 20 22 4
Research Assay—Chef-Ready
(Cat. No. A32928)
Oncomine™ Myeloid Research Assay – 17 (DNA) N/A 4
Chef‑Ready (Cat. No. A36941) 25–28 (RNA)
Oncomine™ Comprehensive Assay v3C 15 (DNA) 18 (DNA) 8 (DNA)
(Cat. No. A35806) 28 (RNA) 31 (RNA) 4 (RNA)
Oncomine™ Comprehensive Assay Plus 13 (DNA) 16 (DNA) 16 (DNA)
– Automated Library Preparation 23 (RNA) 29 (RNA) 4 (RNA)
(Cat. No. A49667)[2]

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 35
 Chapter 4 Run the Ion Chef™ System
4 Start the Ion Chef™ run

Recommended number of amplification cycles for Ion AmpliSeq and Oncomine Research
Assay Chef-Ready panels (continued)
Recommended number of
amplification cycles Anneal/
Assay (10 ng DNA/RNA, 3,000 copies) extension time
High quality Low quality (minutes)
DNA/RNA DNA/RNA[1]
Oncomine™ Childhood Cancer 16 (DNA) 19 (DNA) 8 (DNA)
Research Assay–Chef-Ready 28 (RNA) 31 (RNA) 4 (RNA)
(Cat. No. A36486)
Oncomine™ Focus Assay, Chef‑Ready 16 (DNA) 19 (DNA) 8 (DNA)
(Cat. No. A42008) 28 (RNA) 31 (RNA) 4 (RNA)
[1] FFPE DNA/RNA or cfDNA/RNA
[2] Due to the disparity in the required number of target amplification cycles for high quality and FFPE RNA we do NOT
recommend running both high quality and FFPE samples on the same plate using the same input amount. If preparing a
positive control (high quality) along with FFPE RNA samples, reduce the positive control sample input to 2 ng and use the
FFPE cycling parameters.

The number of amplification cycles that are recommended in the preceding tables are based on
10 ng DNA/RNA input. Adjust the number of amplification cycles for lower or higher DNA/RNA
input:

Amount of DNA/RNA starting material Adjustment to number of amplification cycles

1 ng (300 copies) +3

10 ng (3,000 copies) 0

100 ng (30,000 copies) –3

8. Touch Start Run.

9. After approximately 7–11 hours, return to the Ion Chef™ Instrument. On the Run Complete screen,
touch Next to proceed to the unloading and cleaning steps.

36 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 4 Run the Ion Chef™ System
Start the Ion Chef™ run 4

IMPORTANT! After a run, the Ion Chef™ Instrument holds the barcoded libraries in the tube that
is loaded in Position D of the Reagents cartridge. If you are using the Oncomine™ Comprehensive
Assay Plus, both positions C and D of the Reagents Cartridge contain the barcoded libraries. To
avoid fluid loss due to evaporation, remove and cap the tube of combined barcoded libraries as
soon as possible after run completion. Do not leave the tube in the instrument longer than 24
hours after the start of the run. After 24 hours from the start of the run, the instrument chiller stops
actively cooling, and the sample is held at 27°C.

Note: Touch Run Details to view run information.

You can also monitor the run on the Torrent Browser by selecting Monitor4Ion Chef, and viewing
the Library Prep Progress bar and Library Prep Status associated with your Sample Set.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 37
 Chapter 4 Run the Ion Chef™ System
4 Unload the Ion Chef™ Instrument

Unload the Ion Chef™ Instrument

Remove the tube containing the combined library at approximately 100-pM concentration from Position
D of the Reagents cartridge, and remove used consumables from the instrument from the indicated
stations. If you are using the Oncomine™ Comprehensive Assay Plus, also remove the tube from
Position C of the Reagents cartridge.

3 5

4
2

1 Solutions station
2 Reagents station
3 New Pipette Tip station: move the empty Tip Cartridge to the Used Pipette Tip station
4 Thermal cycler sample block
5 Used Pipette Tip station
6 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 mechanism engages.

IMPORTANT! Lift the door from the center.

1 Center of door

38 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 4 Run the Ion Chef™ System
Unload the Ion Chef™ Instrument 4

2. Remove the Ion AmpliSeq™ Chef Reagents DL8 cartridge. Remove and cap the combined library
tube from Position D, then discard the cartridge.
IMPORTANT! If you are using the Oncomine™ Comprehensive Assay Plus – Automated
Library Preparation, cap both combined library tubes from Positions C and D. The Oncomine™
Comprehensive Assay Plus – Automated Library Preparation combines 4 libraries per pool, A1–A4
in Position C and A5–A8 in position D.

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

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

5. Remove, then discard the box of used pipette tips from the Used Pipette Tip station.

Note:
· Handle the disposable reservoir in the Used Tip station with care. During the run, liquid waste
collects in the reservoir.
· Appropriately discard the liquid waste by tipping the reservoir on the corner with the slot and
pouring the waste into a waste container through that slot.
· Do not reuse the Used pipette tip rack.

6. Move the empty Tip Cartridge L8 from the New Pipette Tip station to the Used Pipette 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 to use in template
preparation. Store unused portions of combined libraries at 4°C to 8°C for up to 1 month. For longer-
term storage, store at –30°C to –10°C. See the appropriate Ion Chef™ or Ion OneTouch™ 2 template kit
user guide for detailed instructions for template preparation.

(Optional) Combine libraries from multiple Ion Chef™ runs

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

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 39
 Chapter 4 Run the Ion Chef™ System
4 Clean the Ion Chef™ Instrument

Clean the Ion Chef™ Instrument

About the cleaning protocol
The Ion Chef™ System includes an automated cleaning function that must be performed following every
run. The cleaning routine is initiated from the Ion Chef™ Instrument touchscreen and is designed to
minimize potential contamination. During the routine, the instrument irradiates the deck with ultraviolet
light for 1 minute after all consumables have been removed from the instrument.

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

Clean the Ion Chef™ Instrument

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

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

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

Note: You can also clean the instrument at any time starting from the home touchscreen. Tap
Settings, then tap Clean Ion Chef.

40 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 4 Run the Ion Chef™ System
Clean the Ion Chef™ Instrument 4

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

4. With the door closed, tap Start.

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

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

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 41
 5 Templating, sequencing and data
analysis

Create a Planned Run with Sample Sets

You must create a Sample Set before you plan an instrument run. See “Create a Sample Set—Imported
file” on page 24.

IMPORTANT! If you are using the Oncomine™ Comprehensive Assay Plus, follow the instructions
for creating a planned run in the Oncomine™ Comprehensive Assay Plus User Guide (Pub. No.
MAN0018490).

Note that a Sample Set that uses an Ion AmpliSeq™ library preparation kit must also use the
corresponding barcode kit that comes with the library preparation kit.

1. In the Plan tab, click Samples.

2. In the Sample Sets screen, select one or more Sample Sets to add to the Planned Run.
• To plan a run using a single Sample Set, click (Actions) 4Plan Run in the row of the
Sample Set.

42 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 5 Templating, sequencing and data analysis
Create a Planned Run with Sample Sets 5

• To plan a run using multiple Sample Sets, select the checkboxes next to the Sample Sets that
you want to add to the Planned Run, then click Plan Run.

IMPORTANT! Ensure that all selected Sample Sets use the same barcode kit. To verify the
barcode kit that is used, expand the Sample Set entry to view its details.

The Select a Run Template to apply to this experiment dialog box lists Planned Run templates
that support your Sample Set.

3. In the Plan Run from Sample Set dialog box, select a run template to use for the experiment, then
click Plan Run.
If you do not see the template that you are looking for, select Show All Templates, then look again
for the template.
The Create Plan workflow opens to the Barcoding step with the Sample Sets that you selected:

4. In the Barcoding step in the workflow bar, enter or select the required information. Depending on
your sequencing application, options can vary.

Option Description
Analysis Parameters Select Default to accept default analysis parameter settings
(recommended).
Advanced users can customize analysis parameters by selecting Custom,
then editing appropriate analysis options. For more information, see the
help system for your version of Torrent Suite™ Software.
Reference Library Select the reference library file that is appropriate for your sample.
Depending on your application, you may have to select separate DNA,
RNA, and fusions reference library files.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 43
 Chapter 5 Templating, sequencing and data analysis
5 Create a Planned Run with Sample Sets

(continued)
Option Description
Target Regions Select the target regions BED file appropriate for your sample. Depending
on your application, you may have to select separate DNA and fusions
target regions files.
Ensure that you are using the current BED or VCF files.
Hotspots Select the hotspots (BED or VCF) file appropriate for your sample.
Ensure that you are using the current BED or VCF files.

5. Select the Use same reference & BED files for all barcodes checkbox if you are using the same
reference, target regions, and hotspots files across all barcoded samples in the Planned Run.
If you are using different reference and/or BED files for one or more of your barcoded samples,
deselect the Use same reference & BED files for all barcodes checkbox.

6. In Sample Tube Label, scan or enter the barcode of the Ion Chef™ sample tubes to be used in the
run.

7. In Chip Barcode, scan or enter the barcode that is printed on the chip that is used for this run.

8. Complete the samples table.

• You can save the samples table to a CSV file, fill out all required sample information, and then
upload the samples table to populate the Samples Table automatically.
a. Click Save Samples Table to save the CSV file to your computer.
b. Edit the CSV file by entering all required sample information into the appropriate sample
information columns, then save the CSV file to your computer.
c. Click Load Samples Table, then select an appropriate CSV file containing sample
information specific for this Planned Run.
d. Click Load to populate the samples table with sample information supplied by the CSV
file.
• Alternatively, you can manually enter sample information into the samples table. Depending on
your sequencing application, options can vary.

Options Description
Barcode For barcoded samples, select a barcode.
Sample Name Select a sample that is a part of the selected sample set or sets.
(Required)
Control Type Click the Control Type column heading to expand the column, then select
the control type from the dropdown list.
Sample ID (Optional) Review sample ID information for each sample. To edit Sample
ID, you must edit the Sample Set as described in “Create a Sample Set—
Imported file” on page 24 or “Create a Sample Set manually” on page 25.

44 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Chapter 5 Templating, sequencing and data analysis
Templating, sequencing and data analysis 5

(continued)
Options Description
Sample Description (Optional) Review sample description for each sample. To modify Sample
Description, you must edit the Sample Set as described in “Create a
Sample Set—Imported file” on page 24 or “Create a Sample Set manually”
on page 25.
DNA/Fusions For DNA and fusions application, select DNA or Fusions for each sample.
Reference If using different reference and BED files for one or more samples, click the
Reference column heading to expand the sections, then select reference,
target regions, and hotspots files for each sample.
Annotations Click the Annotations column heading to expand the annotations specific
for your application (for example, cancer type or embryo ID), then complete
the required information.
Ion Reporter workflow Select the Ion Reporter™ Software analysis workflow specific for your run.
If you do not see your analysis workflow, select the Show All Workflows
checkbox in the column heading.
Relation Select sample relationship group.
Gender Select Male, Female, or Unknown.
IR Set ID Set the IR Set ID to the same value for related samples. After file transfer,
in Ion Reporter™ Software, samples with the same IR Set ID are considered
related samples and are launched in the same analysis (for example,
normal sample and its corresponding tumor sample). Do not give unrelated
samples the same IR Set ID value even if the value is zero or blank.

9. Click Next to continue the steps to create the Planned Run.

The software takes you to the next step in the workflow bar. For more information, see the help
system for your version of Torrent Suite™ Software.

10. Click Save & Finish.

The Planned Run is added to the Planned Runs table and can be used in an instrument run.

Templating, sequencing and data analysis

Proceed to templating, sequencing, and data analysis. Follow the instructions for your assay. If your
assay does not have a user guide, contact support for assistance.

Assays User Guide

Ion AmpliSeq™ automated library preparation on the Ion Chef™ System
Ion AmpliSeq™ Transcriptome Human Gene Expression Ion AmpliSeq™ Transcriptome Human Gene Expression
Panel, Chef-Ready Kit (Cat. No. A31446) Kit User Guide (Pub. No. MAN0010742)
Ion AmpliSeq™ Transcriptome Mouse Gene Expression Ion AmpliSeq™ Transcriptome Mouse Gene Expression
Panel, Chef-Ready Kit (Cat. No. A36412) Kit User Guide (Pub. No. MAN0017343)

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 45
 Chapter 5 Templating, sequencing and data analysis
5 Templating, sequencing and data analysis

(continued)
Assays User Guide
™ ™
Oncomine Research Assay automated library preparation on the Ion Chef System
Oncomine™ BRCA Research Assay Chef-Ready Library Oncomine™ BRCA Research Assay User Guide
Preparation (Cat. No. A32841) (Pub. No. MAN0014634)
Oncomine™ Tumor Mutation Load Assay – Chef‑Ready Oncomine™ Tumor Mutation Load Assay User Guide
Library Preparation (Cat. No. A37910) (Pub. No. MAN0017042)
Oncomine™ Immune Response Research Assay Oncomine™ Immune Response Research Assay User
(Cat. No. A32928) Guide (Pub. No. MAN0015867)
Oncomine™ Myeloid Research Assay – Chef‑Ready Oncomine™ Myeloid Research Assay User Guide
Library Preparation (Cat. No. A36941) (Pub. No. MAN0016113)
Oncomine™ Comprehensive Assay v3C Oncomine™ Comprehensive Assay v3 User Guide
(Cat. No. A35806) (Pub. No. MAN0015885)
Oncomine™ Comprehensive Assay Plus – Automated Oncomine™ Comprehensive Assay Plus User Guide
Library Preparation (Cat. No. A49667) (Pub. No. MAN0018490)
Oncomine™ Childhood Cancer Research Assay–Chef- Oncomine™ Childhood Cancer Research Assay User
Ready (Cat. No. A36486) Guide (Pub. No. MAN0017117)
Oncomine™ Focus Assay, Chef‑Ready (Cat. No. A42008) Oncomine™ Focus Assay, Part I: Library Preparation User
Guide (Pub. No. MAN0013530)

46 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 A Tips and troubleshooting

View troubleshooting and FAQs online

Visit our online Support Centers and FAQ database for tips and tricks for conducting your experiment,
troubleshooting information, and FAQs. The online FAQ database is frequently updated to ensure
accurate and thorough content.
• For the Next–Generation Sequencing Support Center: http://thermofisher.com/ngssupport
• For general Ion AmpliSeq™ library FAQs: http://thermofisher.com/4475345faqs
• To browse the FAQ database and search using keywords: thermofisher.com/faqs

Tips
• Up to 100 ng of DNA or RNA can be used in the standard protocol if the sample quality or quantity
is variable. Subtract up to 3 cycles from the target amplification if uniformity suffers.
• When using the Ion AmpliSeq™ Direct FFPE DNA Kit, samples may be processed in Eppendorf™
DNA LoBind™ Microcentrifuge Tubes before target amplification.
• When using the Ion AmpliSeq™ Comprehensive Cancer Panel, the 4 primer pools provided can be
collapsed into 2 primer pools for library preparation on the Ion 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.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 47
 Appendix A Tips and troubleshooting
A Ion Chef™ System setup and operation

Ion Chef™ System setup and operation

Observation Possible cause Recommended action
Instrument does not display Multiple causes are possible. 1. Power off the instrument, wait
the home screen when 30 seconds, then power on the
powered on instrument.
2. If the instrument fails again, contact
Technical Support.
The instrument door cannot be An obstruction was present on Remove the obstruction blocking the door,
opened or around the door mechanism. then operate the instrument normally.
A hardware or software error Contact Technical Support to report the
occurred. problem and for further assistance.
The instrument does not start a The instrument encountered a 1. Confirm that the touchscreen does not
run Deck Scan error (one or more display any Deck Scan warnings. If
consumables are absent or alarms are present, note the one or more
loaded improperly). errors displayed, replace the missing
consumable as directed, tap No when
prompted, then tap Next to cancel the
run. After returning to the home screen,
restart the run.
2. If the error persists, ensure that:
• All cartridges and PCR plate are
loaded correctly and are level on the
instrument deck.
• The barcodes of the 4 flagged tubes
in Positions A–D of the Reagents
cartridge are visible and positioned
correctly.
• All tubes are both present and
uncapped in the Reagents cartridge.
3. If the error persists after you check the
consumables on the instrument deck, do
one of the following:
• If you are confident that the
Ion Chef™ Instrument is set up
correctly, and you are comfortable
disregarding the warnings, tap YES
following Deck Scan to proceed with
the run.
• If the instrument cannot start the
run, contact Technical Support for
further help.
The instrument encountered an 1. Record the error that is displayed on the
internal error. instrument display, then tap OK.
2. Contact Technical Support to report the
problem and for further help.

48 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix A Tips and troubleshooting
Ion Chef™ System setup and operation A

Observation Possible cause Recommended action

The instrument stops during a The instrument encountered an 1. Record the error displayed on the
run internal error. instrument display, then tap OK.
2. Contact Technical Support to report the
problem and for further assistance.
A consumable was improperly Ensure that all consumables are loaded
loaded. according to the instructions provided.
Instrument displays one or The instrument detected one or After the instrument completes the run,
more alerts during a run more problems during the run. contact Technical Support. If possible, capture
an image of the alert or error message to help
troubleshoot.

IMPORTANT! The detected problem might

impact the performance of the sequencing
run.
• Network connection to the 1. Tap the Instrument status button to view
server was interrupted. the alert(s).
• User name or password 2. In the Instrument status screen, confirm
was incorrect. that the name of the Ion Torrent™ Server
connection is red.
3. Contact your network administrator to
confirm that:
• The Ion Torrent™ Server can be
accessed from the network port
used by the Ion Chef™ Instrument.
If not, troubleshoot the network
connection.
• The user name and password
used by the Ion Chef™ Instrument
are valid. If not, contact the
server administrator to renew the
credentials.
4. If the alert persists, capture an image of
the alert or error message, if possible, to
help troubleshoot, then contact Technical
Support for further assistance.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 49
 B Ion AmpliSeq™ panels

Oncomine™ tumor specific panels

The Ion Torrent™ Oncomine™ tumor specific panel designs are small curated panels with verified
performance that complement the on-market Oncomine™ assays and provide an end-to-end solution
for research of specific tumors such as bladder, prostate, melanoma, and others from FFPE tissue
samples.
The Oncomine™ tumor specific panels provide the following benefits.
• Small tumor specific panels with relevant content for research
• End-to-end workflow support that includes bioinformatics and reporting
• Low sample input requirement and robust performance from FFPE tissue samples
• Specialized support for assay verification
• Panel customization

Ten tumor specific panels are available in Ion AmpliSeq™ Designer. The performance of each panel was
verified on oncological research samples. A Copy Number Variant (CNV) baseline is provided with each
panel for data analysis in Ion Reporter™ Software for samples that are sequenced on the Ion 530™ Chip
or Ion 540™ Chip.

Ion AmpliSeq™ On‑Demand Panel designs

Ion AmpliSeq™ On-Demand designs are suitable for common disease research areas and are
customizable at the gene level. The designs include optimized amplicons with verified performance
for germline analysis. You can create designs by selecting from a catalogue of >5,000 pre‑tested genes
or browsing the Ion AmpliSeq™ Designer gene repository by research area of interest.
Each Ion AmpliSeq™ On‑Demand Panel consists of 2 DNA primer pools, where the number of primer
pairs per pool depends on the genes that are selected. The maximum number of genes that can
be selected for design creation is 800. Each Ion AmpliSeq™ On‑Demand panel order is limited to
500 genes or 15,000 amplicons. The minimum number of amplicons per design is 24.
You can add genes that are not in the On-Demand catalog to your panel as a Spike‑in Panel. Spike-in
Panels are high concentration Made-To-Order panels that are used to supplement On‑Demand designs
with genes that are not available On-Demand. Spike‑in Panels are created as 2‑pool designs and are
limited to 123 amplicons per pool.
Ion AmpliSeq™ On-Demand panels are available in 8 or 32 reaction packs for library preparations on
the Ion Chef™ System and 24 or 96 reaction packs for manual library preparation. The panels support
sequencing genomic targets from as little as 10 ng of DNA input using the Ion PGM™ System.

50 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix B Ion AmpliSeq™ panels
Ion AmpliSeq™ Custom panel designs B

Ion AmpliSeq™ Custom panel designs

Custom panel designs are customizable for any genome at the amplicon level. Panels can be designed
against a number of pre-loaded reference genomes, or for any other organism by uploading a FASTA
file reference sequence.
Custom panels are delivered with a few weeks in tubes of pre-pooled, multiplexed primers in ready-to-
use concentrations. You can add 384‑well plates to your order, where each well contains individual
primer pairs in concentrated format.
When ordered in the Tubes Only format, Ion AmpliSeq™ Custom panels contain 750 reactions per pool
for panels with ≤96 amplicons, or 3,000 reactions per pool for panels with >96 amplicons. Panels
ordered in the Tubes Plus 384-Well Plate format contain 2,250 reactions (750 in pools and 1,500 in plate
format) for panels with ≤96 amplicons, or 9,000 reactions (3,000 in pools and 6,000 in plate format) for
panels with >96 amplicons.
Ion AmpliSeq™ Made-to-Order Panels, when ordered in the Tubes Only format, contain 750 reactions
per pool for panels with ≤96 amplicons, or 3,000 reactions per pool for panels with >96 amplicons.
Panels ordered in the Tubes Plus 384-Well Plate format contain 2,250 reactions (750 in pools and 1,500
in plate format) for panels with ≤96 amplicons, or 9,000 reactions (3,000 in pools and 6,000 in plate
format) for panels with >96 amplicons.

Ion AmpliSeq™ Fixed Panels

Ion AmpliSeq™ Fixed Panels are fixed‑design DNA and RNA panels that are created for germline or
somatic analysis. These panels are verified for performance and are suitable for a wide range of
research areas, including inherited diseases, cancer research, human identification, infectious diseases,
pharmacogenomics, developmental disorders, inflammtion and immune response research, exome, and
more. Two types of fixed Ion AmpliSeq™ panels are available - Community Panels and Ready‑to‑Use
Panels.
Ion AmpliSeq™ Community Panels that are designed with input from leading disease researchers and
are available to ship withing a few weeks. Ion AmpliSeq™ Ready‑to‑Use Panels are in‑stock panels that
are design by Thermo Fisher Scientific researchers and are ready to ship immediately.
When ordered in the Tubes Only format, Ion AmpliSeq™ Fixed Panels contain 750 reactions per pool for
panels with ≤96 amplicons, or 3,000 reactions per pool for panels with >96 amplicons. Panels ordered
in the Tubes Plus 384-Well Plate format contain 2,250 reactions (750 in pools and 1,500 in plate format)
for panels with ≤96 amplicons, or 9,000 reactions (3,000 in pools and 6,000 in plate format) for panels
with >96 amplicons.
Ion AmpliSeq™ community panels, designed with input from leading disease researchers and verified for
performance, provide targeted sequencing for your research in cancer, inherited disease, and microbial
and infectious disease. These panels are fully customizable to fit your unique project requirements and
advance clinical research, giving you the flexibility to add or remove genes and amplicons.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 51
 C Supplemental procedures

■ Reverse transcribe RNA using the SuperScript™ VILO™ cDNA Synthesis Kit . . . . . . . . . . . . . . . . . 52
■ (Optional) Qubit™ Fluorometer: Quantify the FFPE DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
■ Create a samples file from a template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

Reverse transcribe RNA using the SuperScript™ VILO™

cDNA Synthesis Kit
Reverse transcription reactions can be performed with the SuperScript™ VILO™ cDNA Synthesis Kit,
which contains the 10X SuperScript™ III Enzyme Mix. This enzyme mix requires that you prepare a
reverse transcription Master Mix with 5X VILO™ Reaction 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-heat at 80°C for
10 minutes, then cool to room temperature.

2. Warm the 5X VILO™ Reaction Mix to room temperature, then vortex to mix.

3. If there is any visible precipitate in the 5X VILO™ Reaction Mix, vortex further until the mix is
completely resuspended.

4. Prepare a reverse transcription Master Mix. For 8 samples, combine the following components 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™ Reaction Mix (blue 3 µL 27 µL
cap)
10X SuperScript™ III Enzyme Mix 1.5 µL 13.5 µL
(black cap)
[1] One reaction added as overage to compensate for pipetting error.

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

6. Pipet 4.5 µL of the reverse transcription Master Mix into wells A1 to H1 of the plate.

52 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix C Supplemental procedures
Reverse transcribe RNA using the SuperScript™ VILO™ cDNA Synthesis Kit C

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

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

E
F
G

1 2

1 Each column 1 well contains 4.5 µL reverse transcription Master Mix, and 10.5 µL of diluted RNA sample
(0.95 ng/µL, 10 ng total).
2 Each column 6 well contains a dried-down IonCode™ barcode. The lowest barcode number is in A6, and the
highest is in H6. All appear light blue in the actual plates.

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

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

9. Seal the plate with a MicroAmp™ Clear Adhesive Film, vortex thoroughly, then centrifuge briefly to
collect droplets. Alternatively, mix by pipetting at least 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 the barcode label. If
the barcode label becomes damaged, you can override the error during Deck Scan.

10. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal cycler, 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 the thermal cycler. For
longer term, store at –30°C to –10°C.

11. Remove the plate from the thermal cycler, then gently tap the plate to collect any condensate.
Alternatively, centrifuge briefly to collect droplets.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 53
 Appendix C Supplemental procedures
C (Optional) Qubit™ Fluorometer: Quantify the FFPE DNA

Proceed to “Thaw the reagents and prepare the instrument” on page 23.

(Optional) Qubit™ Fluorometer: Quantify the FFPE DNA

When using the Ion AmpliSeq™ Direct FFPE DNA Kit, the DNA concentration can be estimated using a
Qubit™ Fluorometer and the Qubit™ dsDNA HS Assay 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 and samples. The Qubit™
dsDNA HS Assay requires 2 standards.

2. Prepare sufficient Qubit™ working solution for all samples and standards by diluting 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) of working 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. (Qubit™ 2.0 Fluorometer only.) Calculate the concentration of the undiluted sample by multiplying
by the dilution factor. Alternatively, use the Calculate Stock Conc. feature on your instrument.

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

Create a samples file from a template

If you do not already have a samples file on your computer to use to import samples from a CSV file,
then you can download a samples template and use it to create a samples file.

1. In the Plan tab, click Samples, then click Import Samples from File.

2. In step 1 of the Import Samples section, click Sample File Format to download a sample CSV
template.
The sample file format CSV contains the version of the CSV file in the top row, and sample
attributes in separate columns.

3. To create a new CSV file, copy and paste the contents of your existing sample CSV file into the
new file format.

54 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix C Supplemental procedures
Create a samples file from a template C

4. Open the CSV template and enter sample information into the cells.
For information on how to define the samples, see “Sample attributes” on page 55.

5. Save the file to your computer so that is available for use to create a Sample Set.

Sample attributes
When you create a Sample Set, you enter sample attributes into the Torrent Suite™ Software, whether
you enter attributes manually or by importing samples using a CSV file. Most of the sample information
is optional except for Sample Name. However, some information is required if you transfer data to
Ion Reporter™ Software, and those attributes required by Ion Reporter™ Software are indicated in the
following table.

Attribute Description

Sample Name The unique name of the sample.

(Required) Use any combination of alphanumeric characters, plus spaces, periods (.),
hyphens (-), and underscores (_).
This attribute is used by Ion Reporter™ Software.
A 255‑character limit must be followed for Ion Reporter™ Software name
validation. If you are using Ion Reporter™ Software, and the actual sample name
already exists in that software, a string such as _v1 or _v2, and so on, is added to
the sample name.

Sample External ID (Optional) If you manage samples in an external system, for example, a
Laboratory Information Management System (LIMS), enter the identifier from that
system.

Barcode Kit The name of the barcode kit used to make a library from the sample. The same
barcode kit must be used for all samples in a Sample Set.

Barcode The name of the specific barcode in the selected barcode kit. Assign a unique
barcode to each sample in a Sample Set.

Sample ID A unique identification code (SampleID) for each barcode in a sample. This
helps to track samples or possibly identify misassignment between samples
and barcodes in a sequencing run. The SampleID is passed to Ion Reporter™
Software.
If you manage samples in an external system (for example, a LIMS), you can use
the identifier from that system.
This attribute cannot be changed.
Note: This attribute is optional in Torrent Suite™ Software. However, this value
can be required for planned runs that include automatic upload of data to Ion
Reporter™ Software.

Control Type The control type used when preparing the sample. If blank, the sample is not a
control type sample.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 55
 Appendix C Supplemental procedures
C Create a samples file from a template

(continued)

Attribute Description

Basic Annotations

Description Typically, one or two sentences that describe the sample.

Nucleotide Type • Unspecified

• DNA
• RNA
• TNA
• Fusions

Sample Source The source from which the sample is extracted.

• Blood
• FFPE
• Other

Panel Pool Type Reserved for future use.

• Dual Pool
• Single Pool

Gender The biological gender of the sample.

This attribute is used by Ion Reporter™ Software.
• Female
• Male
• Unknown

IMPORTANT! If you are using Ion Reporter™ Software, do not leave this
blank. Select Unknown if the gender is not known. Several analysis workflows
in Ion Reporter™ Software, for example, copy number variation detection and
genetic disease research, are limited when the gender is not known. The
analysis workflows can return unexpected results when the gender is incorrectly
specified for a sample.

56 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix C Supplemental procedures
Create a samples file from a template C

(continued)

Attribute Description

Type The relationship type for this sample. Type is used in conjunction with
Relationship Group, described below. For example, a relationship group can
contain two samples, one with a type Tumor and another with a type Normal.
The following sample relationships are supported by Ion Reporter™ Software:
• Control
• Father
• Mother
• Normal
• Sample
• Self—Use Self for both a single sample and for the proband sample in a trio.
A single sample is not related to other samples and is analyzed by itself.
• Tumor

Relationship Group Use Relationship Group to designate a group of multiple related samples within
the same Sample Set. For example, DNA and RNA samples from the same
sample would have the same Relationship Group number.
Use a whole number to define a sample as part of a relationship group. It is used
in conjunction with Type. For example, a Sample Set can contain 6 samples,
consisting of 3 groups of 2 related samples each (of types Tumor and Normal). In
this case, you would designate the two samples in each group as part of group
1, 2, or 3. This is identical to the Set ID in the IonReporterUploader plugin.

Extra Annotations (used for specialized applications, such as preimplantation genetic screening (PGS)
research or oncology research)

Sample Collection Date The date that the blood sample was drawn.

Sample Receipt Date The date that the laboratory received the blood sample.

Cancer Type The type of cancer that is present in the sample.

Population The super population code assignment for a sample, as defined by the
human 1000 genomes project ( www.internationalgenome.org/faq/which-
populations-are-part-your-study/). The population is relevant to the analysis
of samples by the TCRB-LR assay workflow in Ion Reporter™ Software. The
TCRB-LR analysis workflow produces a haplotype group assignment for samples
having a population attribute of "European".

Mouse Strains The name of a mouse strain. Choose from a select number of the most common
strains. In Ion Reporter™ Software 5.12, the selected mouse strain will not affect
Ion Reporter™ Software analysis workflows.

Cellularity % The percentage of tumor cells in the sample. This is a whole number between 1
and 100.

Biopsy Days The timepoint post-fertilization at which the biopsy was taken from an embryo.
This is a whole number.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 57
 Appendix C Supplemental procedures
C Create a samples file from a template

(continued)

Attribute Description

Cell Number The cell count of the biopsied material.

Couple ID An identifier for the couple. For use with the Reproductive research application.

Embryo ID An identifier for the embryo. For use with the Reproductive research application.

User-defined Attributes

<user defined> If you create additional sample attributes, each attribute is listed here and in
the CSV file. Attributes that are marked as mandatory must be entered for each
sample. If you create an attribute of type integer, only numeric characters (whole
numbers) can be entered for that attribute.

58 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 D Strategies for combining Ion
AmpliSeq™ libraries

■ Combine barcoded libraries from multiple runs for sequencing on a single Ion chip . . . . . . . . . . . 59
■ Combine libraries prepared using different panels for equal coverage . . . . . . . . . . . . . . . . . . . . . . . 60
■ Combine unequal volumes of libraries for varying depth of coverage . . . . . . . . . . . . . . . . . . . . . . . . 61
■ Ion Chip capacities for Ion AmpliSeq™ DNA libraries sequenced at equal depths . . . . . . . . . . . . . 62
■ Ion Chip capacities for Ion AmpliSeq™ RNA libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

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

Combine barcoded libraries from multiple runs for

sequencing on a single Ion chip
The Ion AmpliSeq™ Kit for Chef DL8 contains reagents and materials sufficient to prepare up to 32
uniquely barcoded libraries. If the same Ion AmpliSeq™ panel (or two panels with the same number
of amplicons) was used over multiple runs with different barcodes, the combined libraries can be
super-pooled by adding equal volumes of each. See “Ion Chip capacities for Ion AmpliSeq™ DNA
libraries sequenced at equal depths” on page 62 and “Ion Chip capacities for Ion AmpliSeq™ RNA
libraries” on page 63 to determine how many libraries you can sequence on various 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 libraries prepared in multiple
Ion AmpliSeq™ for Chef runs are super-pooled.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 59
 Appendix D Strategies for combining Ion AmpliSeq™ libraries
D Combine libraries prepared using different panels for equal coverage

Combine libraries prepared using different panels for equal

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

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

2. Combine the libraries using the following formula:

Number of primer pairs in Panel 1

● Volume (μL) of library from Panel 1 = 100 μL
Total number of primer pairs in Panels 1 and 2

Number of primer pairs in Panel 2

● Volume (μL) of library from Panel 2 = 100 μL
Total number of primer pairs in Panels 1 and 2

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

60 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix D Strategies for combining Ion AmpliSeq™ libraries
Combine unequal volumes of libraries for varying depth of coverage D

Combine unequal volumes of libraries for varying depth of

coverage
Ion AmpliSeq™ libraries with unique barcodes may be super-pooled to produce disproportionate
numbers of reads. Use this strategy for combining DNA and RNA libraries 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, while an average depth of coverage at
~2500X may be preferred for the tumor sample. In this 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
sample run with 90 µL of the combined library from a tumor sample run.

Fractional
Sample
volume/reads

Combined libraries from

0.1
Chef run #1 (normal)

Combined libraries from

0.9
Chef run #2 (tumor)

Sum 1.0

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 61
 Appendix D Strategies for combining Ion AmpliSeq™ libraries
D Ion Chip capacities for Ion AmpliSeq™ DNA libraries sequenced at equal depths

Ion Chip capacities for Ion AmpliSeq™ DNA libraries

sequenced at equal depths
The number of combined libraries that can be accommodated in a single sequencing run depends on
the chip, the balance of barcoded library concentration, and the coverage required.
For a given chip, as the number of amplicons increases, the number of libraries that can be
accommodated per sequencing run decreases. This relationship is shown in the following table. The
numbers in the following table serve as a guide for approximate capacities. As the number of libraries
per chip increases, it becomes more difficult to balance the reads between libraries. In addition, libraries
from FFPE tissue tend to produce more variable results. We suggest combining fewer libraries initially
and determining real limits empirically.

Ion 316™ Ion 318™ Ion PI™

Chip Ion 314™[1] Ion 530™ Ion 550™
Ion 510™ Ion 520™ Ion 540™

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

Expected 95% 95% 95% 95% 95% 95% 95% 95% 95% 95% 95% 95%
coverage >30X >500X >30X >500X >30X >500X >30X >500X >30X >500X >30X >500X

Number of
amplicons per Approximate number of libraries per chip
library

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

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

49–96 26 2 84 8 152 14 >384 48 >384 152 >384 243

97–192 14 1 48 4 84 8 272 26 >384 84 >384 134

193–384 8 — 26 2 48 4 152 14 >384 48 >384 77

385–768 4 — 14 1 26 2 84 8 272 26 >384 41

769–1,536 2 — 8 — 14 1 48 4 152 14 243 22

1,537–3,072 1 — 4 — 8 — 26 2 84 8 134 13

3,073–6,144 — — 2 — 4 — 14 1 48 4 77 7

6,145–12,288 — — 1 — 2 — 8 — 26 2 41 3

12,289–24,576 — — — — 1 — 4 — 14 1 22 2

24,577–49,152 — — — — — — 2 — 8 — 13 1
[1] Discontinued, but still supported.

62 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix D Strategies for combining Ion AmpliSeq™ libraries
Ion Chip capacities for Ion AmpliSeq™ RNA libraries D

Ion Chip capacities for Ion AmpliSeq™ RNA libraries

If there is a user guide for your RNA panel, follow the recommendations in the user guide for your panel.
The following guidelines can be used for panels without a user guide. These recommendations serve
as suggestions and the actual capacity to multiplex libraries is determined by the expression levels of
the genes included in your Ion AmpliSeq™ RNA panel. The expression levels of the individual genes
can vary by input RNA type. We suggest using the formula for new panels and determining actual
multiplexing limits empirically.
• We recommend that you plan for an average of 5,000 reads per amplicon for an Ion AmpliSeq™
RNA library targeting 1–200 genes. The actual sequencing depth that is required depends on
the expression levels of the gene targets in your sample RNA, so scale the sequencing depth to
accommodate your sample type and research needs.
• For panels containing fusion detection primer pairs, higher library multiplexing is possible because
most targets are not present, and therefore do not create library molecules. For most fusion
detection assays, only ~250,000 reads per library are required.
• Use the following formula and chip capacity table to provide initial guidance for multiplexing
RNA‑derived gene expression libraries on Ion sequencing chips.
Chip capacity in reads
Number of libraries sequenced per chip =
(Sequencing depth) x (Number of primer pairs)

Ion Chip

Ion 318™/ Ion PI™/

Ion 314™ Ion 316™ Ion 510™ Ion 530™ Ion 550™
Ion 520™ Ion 540™
Chip[1] Chip Chip Chip Chip
Chip Chip

Chip 0.3−0.5 1−2 2–3 3−5 15−20 60−80 100–130

capacity in
reads (M)
[1] Discontinued, but still supported.

Example:
Chip capacity of Ion 540™ Chip = ~60,000,000 reads
Sequencing depth desired = 5,000 reads per amplicon
Number of primer pairs = 100
60,000,000 / (5,000 × 100) = 120 libraries per Ion 540™ Chip

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 63
 E Ion AmpliSeq™ Made-to-Order
Panels

Each Ion AmpliSeq™ Made-to-Order Panel order includes primer pools at the standard 2X or 5X
concentration, in some cases, 384-well plates containing all the individual primer pairs. Each plate
well contains the forward and reverse primer in Low 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 a pool do not
overlap. When a panel with a new footprint is created, the designed BED file should be modified
accordingly.

Prepare primer pools from plates

To create a primer pool from 384‑well plates, combine and dilute the desired primer pairs to the
appropriate concentration (per primer):
• For panels with up to 96 primer pairs per pool, combine and dilute to 1,000 nM per primer (5X,
1,000 nM).
• For panels with 97–1,228 primer pairs per pool, combine and dilute to 250 nM per primer (5X,
250 nM).
• For panels with 1,229–3,072 primer pairs per pool, combine and dilute to 100 nM per primer (2X,
100 nM).

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.

64 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix E Ion AmpliSeq™ Made-to-Order Panels
Prepare primer pools from plates E

3. Add Low TE (10 mM Tris, pH 8.0; 0.1 mM EDTA) to the tube to the appropriate final volume shown
in the following table:
To make primer pools Action
5X, 1,000 nM Add Low TE to a final volume of 0.614 mL[1].
(12−96 primer pairs)
5X, 250 nM Add Low TE to a final volume of 2.456 mL [2].
(97−1,228 primer pairs)
2X, 100 nM Add Low TE to a final volume of 6.140 mL.
(1,229−3,072 primer pairs)
50X spike-in panel, 2,500 nM Add Low TE to a final volume of 0.246 mL.
(1−123 primer pairs)
[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.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 65
 F Expand any Ion AmpliSeq™ panel by
adding a spike‑in panel

An Ion AmpliSeq™ On‑Demand Panel can be modified by using a spike‑in panel. Spike‑in panels enable
addition of new content, control assays, and improvements in coverage uniformity (for example, by
adding the same primer pair to two or more primer pools).

1. If needed, prepare a 50x (2500 nM) spike-in panel from individual primer pairs using the procedure
in Appendix E, “Ion AmpliSeq™ Made-to-Order Panels”.

2. Add the spike-in panel (at 2,500 nM) to a primary Ion AmpliSeq™ panel using the following
guidelines.

Number of primer pairs/pool Target Concentration

Instructions
Primary Panel Spike-in Combined (Combined)

Chef-Ready primary panels

Add 6.4 µL of the spike-in panel to the Chef-Ready panel tube.
All other primary panels
Less than 96 Up to 123 Less than 96 2X:400 nM Add 16 µL of the spike‑in panel per
100 µL of the primary panel
Less than 96 Up to 123 More than 96 2X:100 nM Dilute parent panel from 400 nM to
100 nM, then add 4 µL spike-in panel
per 100 µL of primary panel
Greater than 96 Up to 123 More than 96 2X:100 nM Add 4 µL spike-in panel per 100 µL
primary panel

3. Mix thoroughly by vortexing, then centrifuge briefly to collect contents.

The modified primer pools are ready to use. No adjustment of volume is necessary.

Examples of spike-in volumes for different categories of parent and spike‑in panel

Volumes for Final

Number of primer pairs/pool Concentration (nM)
spiking (µL) concentration (nM)

Target
Parent Spike-in Combined Parent Spike-in Parent Spike-in Parent Spike-in
(combined)

50 20 70 400 2,500 400 160 25.6 345 345

80 60 140 100 2,500 100 160 6.4 96.2 96.2

(diluted)

66 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix F Expand any Ion AmpliSeq™ panel by adding a spike‑in panel
Prepare primer pools from plates F

Examples of spike-in volumes for different categories of parent and spike‑in panel (continued)

Volumes for Final

Number of primer pairs/pool Concentration (nM)
spiking (µL) concentration (nM)

Target
Parent Spike-in Combined Parent Spike-in Parent Spike-in Parent Spike-in
(combined)

3,000 120 3,120 100 2,500 98.5 160 6.4 96.2 96.2

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 67
 G Ion AmpliSeq™ Sample ID Panel

Using the Ion AmpliSeq™ Sample ID Panel

If your panel includes the Ion AmpliSeq™ Sample ID Panel, do not add additional panel. For additional
details, see the user guide for your assay.
The Ion AmpliSeq™ Sample ID Panel is an Ion AmpliSeq™ Spike-in panel that is used to add amplicons
to create a unique identifier for human gDNA samples. It is comprised of 9 primer pairs at a 20X
concentration (1000 nM per primer). If you are using the Ion AmpliSeq™ Kit for Chef DL8, use the
following procedure to create a sample signature in your panel.

1. For addition to a primary panel with greater than 96 primer pairs per pool, add 15 µL of the Ion
AmpliSeq™ Sample ID Panel and 135 µL of the primer panel to one or both Primer Pool Tubes.

Note:
· For addition to a primary panel with less than 96 primer pairs per pool, optimization may be
required to obtain the desired read coverage distribution of Sample ID amplicons relative to
primary panel amplicons.
™
· If a primary panel has more than one primer pool, the Ion AmpliSeq Sample ID Panel can be
added to one or all primer pools.
™
· The Ion AmpliSeq Sample ID Panel can be used to match a tumor and normal sample.
However, copy number variations in the tumor sample can distort the allele balance in the
fingerprint.

2. Select the following settings when creating a new Planned Run in the Torrent Browser Planned Run
Wizard:
a. In the Kits step, select the Control Sequence, then choose Ion AmpliSeq Sample ID panel
from the dropdown list.

b. In the Plugins step, select the coverageAnalysis plugin checkbox, then click Configure (see
the help system for your version of Torrent Suite™ Software).

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

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

d. In the Plugins step, select the sampleID plugin. A Sample ID Report is then automatically
generated after the run.

68 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix G Ion AmpliSeq™ Sample ID Panel
Using the Ion AmpliSeq™ Sample ID Panel G

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

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 69
 H Ion AmpliSeq™ RNA ERCC
Companion Panel

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves. Safety Data Sheets (SDSs) are available from
thermofisher.com/support.

Product description
The Ambion™ ERCC RNA Spike-In Mix provides a set of external RNA controls that enables
performance assessment of RNA-Seq libraries in gene expression experiments on Ion Torrent™
sequencing platforms. With the addition of the Ion AmpliSeq™ RNA ERCC Companion Panel to your
Ion AmpliSeq™ RNA panel, dynamic range and sensitivity can be similarly evaluated in Ion AmpliSeq™
RNA libraries that are prepared from RNA samples containing the ERCC RNA Spike-In Mix. Add ERCC
RNA Spike-In Mix to each RNA sample, then amplify targets with an Ion AmpliSeq™ RNA panel plus
Ion AmpliSeq™ RNA ERCC Companion Panel. Sequence the libraries on an Ion PGM™, Ion Proton™,
Ion S5™, Ion S5™ XL, or Ion GeneStudio™ S5 System, then compare the Spike-In Mix data to known
Spike-In Mix concentrations to evaluate the dynamic range and lower limit of detection of the platform.
This user guide provides guidelines for preparing and analyzing Ion AmpliSeq™ RNA libraries with the
ERCC RNA Spike-In Mix and the Ion AmpliSeq™ RNA ERCC Companion Panel.

Note: For use with the Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Cat. Nos. A26325,
A26326, A26327, and A31446) and Ion AmpliSeq™ Transcriptome Mouse Gene Expression Kit (Cat.
Nos. A36553, A36554, A36555, and A36412) only.

Software compatibility
The Ion AmpliSeq™ RNA ERCC Companion Panelis compatible with Torrent Suite™ Software version
5.10 and later and the ERCC_Analysis plugin version 5.10.0.3 or later. Be sure to update your Ion
Torrent™ Server to the latest available version of Torrent Suite™ Software before using this kit.

70 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix H Ion AmpliSeq™ RNA ERCC Companion Panel
Contents and storage H

Contents and storage

The Ion AmpliSeq™ RNA ERCC Companion Panel provides enough reagent for 96 manual reactions or
48 automated reactions.

Contents Amount Storage

Ion AmpliSeq™ RNA ERCC Companion Panel 1 × 96 µL −30°C to −10°C

Note: The Ion AmpliSeq™ RNA ERCC Companion Panel is shipped at ambient temperature. Store as
indicated.

Required materials not supplied

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

Item Source

ERCC RNA Spike-In Mix 4456740

One of the following:

• Ion AmpliSeq™ Transcriptome Human Gene Expression Kit • A26325, A26326, or A26327
™
• Ion AmpliSeq Transcriptome Mouse Gene Expression Kit • A36553, A36554, or A36555
™
• Ion AmpliSeq Transcriptome Human Gene Expression Panel, • A31446
Chef-Ready Kit • A36412
• Ion AmpliSeq™ Transcriptome Mouse Gene Expression Panel,
Chef-Ready Kit

Before you begin

Ensure that version 5.10.0.3 or later of the ERCC_Analysis plugin has been installed on the Torrent
Suite™ Software. This version of the plugin requires Torrent Suite™ Software version 5.10 or later.
• To confirm the version that is installed on the Torrent Suite™ Software, see “Confirm ERCC_Analysis
plugin version” on page 73.
• For instructions for installing the ERCC_Analysis plugin, see “Install the ERCC_Analysis plugin” on
page 73.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 71
 Appendix H Ion AmpliSeq™ RNA ERCC Companion Panel
H Add ERCC RNA Spike-In Mix 1 to RNA samples

Add ERCC RNA Spike-In Mix 1 to RNA samples

IMPORTANT! Ion AmpliSeq™ RNA panel size and content, as well as starting RNA quantity and
quality, affect the percentage of ERCC mapped reads in the final libraries. We recommend using the
following tables as starting points. Further adjustment of amount of ERCC RNA Spike-In Mix added to
each sample may be needed.

1. Determine the amount of ERCC RNA Spike-In Mix 1 to add, using the following table as a
guideline.

Amount of total RNA Volume of diluted ERCC RNA Spike-In Mix 1

10 ng 1 µL (1:5,000 dilution)
20 ng 2 µL (1:5,000 dilution)
50 ng 1 µL (1:1,000 dilution)
100 ng 2 µL (1:1,000 dilution)

2. Prepare the appropriate dilution of ERCC RNA Spike-In Mix 1 needed using the following table.
Scale the volumes accordingly if > 10 μL of the dilution is needed.

Dilution ERCC RNA Spike-In Mix 1 Nuclease-free Water

1:10 1 µL undiluted 9 µL
1:100 1 µL of 1:10 9 µL
1:1,000 1 µL of 1:100 9 µL
1:5,000 2 µL of 1:1,000 8 µL
1:10,000 1 µL of 1:1,000 9 µL
1:50,000 1 µL of 1:5,000 9 µL

Note: Prepare a fresh dilution of the ERCC RNA Spike-In Mix for each procedure. Discard unused
diluted Spike-In Mix.

3. Add the volume of the appropriate ERCC RNA Spike-In Mix 1 dilution, which is determined in
step 1, to each RNA sample. Concentrate the RNA sample containing ERCC RNA Spike-In Mix 1
(centrifugal vacuum concentration recommended), if needed.
Proceed to “Add the Ion AmpliSeq™ RNA ERCC Companion Panel to your RNA panel” on
page 73.

72 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix H Ion AmpliSeq™ RNA ERCC Companion Panel
Add the Ion AmpliSeq™ RNA ERCC Companion Panel to your RNA panel H

Add the Ion AmpliSeq™ RNA ERCC Companion Panel to

your RNA panel
Note: Any Ion AmpliSeq™ Transcriptome Gene Expression panel can be modified through the addition
of the Ion AmpliSeq™ RNA ERCC Companion Panel.

1. Add the Ion AmpliSeq™ RNA ERCC Companion Panel.

Reaction type Amount
Manual reaction Add 1 µL per reaction.
™
Automated (Ion Chef System) Add 8 µL per pool (16 µL per Ion Chef™ Instrument run).

2. Mix thoroughly by vortexing, then centrifuge. The modified primer pools are ready to use.

3. Follow the standard library preparation protocol for Ion AmpliSeq™ RNA library preparation, using
the change in panel volume specified in the table in step 1.
Return to in step 2

Supplemental procedures
Confirm ERCC_Analysis plugin version
If the ERCC_Analysis plugin has not been uploaded to the Torrent Suite™ Software, see “Install the
ERCC_Analysis plugin”.

1. Sign in to the Ion Torrent™ Server connected to your sequencer via Torrent Suite™ Software.

2. Click (Settings)4Plugins

3. Scroll to the ERCC_Analysis plugin to see the installed version.

Install the ERCC_Analysis plugin

The ERCC Companion Panel requires you to upgrade the ERCC_Analysis plugin. You can find this
plugin on the Connect.

1. Sign in to the apps.thermofisher.com.

2. Click the Apps icon ( ).

3. In AppConnect, under Resource Libraries, click Plugins.

4. Select RNA‑Seq in the category bar.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 73
 Appendix H Ion AmpliSeq™ RNA ERCC Companion Panel
H Supplemental procedures

5. Click to download the ERCC_Analysis plugin. Select the checkbox to indicate that you agree to
the terms and conditions, then click Download Plugin.
Either a compressed directory or a debian file that contains the plugin is downloaded to your local
machine.

6. Click (Settings)4Plugins4Install or Upgrade Plugin in Torrent Suite™ Software.

7. Click Select File, then browse to the location where you downloaded the plugin file, select the file,
then click Open.

8. In the Install or Upgrade Plugin dialog box, click Upload and Install.

The plugin is now visible in Torrent Suite™ Software.

74 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 I Safety

WARNING! GENERAL SAFETY. Using this product in a manner not specified in the user
documentation may result in personal injury or damage to the instrument or device. Ensure that
anyone using this product has received instructions in general safety practices for laboratories and
the safety information provided in this document.

· Before using an instrument or device, read and understand the safety information provided in the
user documentation provided by the manufacturer of the instrument or device.
· Before handling chemicals, read and understand all applicable Safety Data Sheets (SDSs) and use
appropriate personal protective equipment (gloves, gowns, eye protection, and so on). To obtain
SDSs, see the “Documentation and Support” section in this document.

Symbols on this instrument

Symbols may be found on the instrument to warn against potential hazards or convey important safety
information. In this document, the hazard symbol is used along with one 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 against unsafe 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 Attention, risque de danger

Consult the manual for further safety Consulter le manuel pour d’autres
information. renseignements de sécurité.

Caution, risk of electrical shock Attention, risque de choc électrique

Caution, hot surface Attention, surface chaude

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 75
 Appendix I Safety
I Symbols on this instrument

(continued)

Symbol English Français

Ultraviolet light Rayonnement ultraviolet

On/Off On/Off (marche/arrêt)

Protective conductor terminal (main ground) Borne de conducteur de protection (mise à la

terre principale)

Do not dispose of this product in unsorted Ne pas éliminer ce produit avec les déchets
municipal waste usuels non soumis au tri sélectif.

CAUTION! To minimize negative MISE EN GARDE ! Pour minimi‐

environmental impact from disposal ser les conséquences négatives sur
of electronic waste, do not l’environnement à la suite de l’éli‐
dispose of electronic waste in mination de déchets électroniques,
unsorted municipal waste. Follow ne pas éliminer ce déchet électro‐
local municipal waste ordinances nique avec les déchets usuels non
for proper disposal provision soumis au tri sélectif. Se conformer
and contact customer service aux ordonnances locales sur les dé‐
for information about responsible chets municipaux pour les disposi‐
disposal options. tions d’élimination et communiquer
avec le service à la clientèle pour
des renseignements sur les options
d’élimination responsable.

Conformity symbols
Conformity mark Description

Indicates conformity with safety requirements for Canada and U.S.A.

Indicates conformity with European Union Low Voltage Directive 2006/95/EC, EMC
Directive 2014/30/EU, and R&TTE Directive 1999/5/EC.

Indicates conformity with Australian standards for electromagnetic compatibility.

76 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix I Safety
Location of safety labels on this instrument I

Location of safety labels on this instrument

Safety information for instruments not manufactured by

Thermo Fisher Scientific
Some of the accessories provided as part of the instrument system are not designed or built by Thermo
Fisher Scientific. Consult the manufacturer's documentation for the information needed for the safe use
of these products.

Instrument safety
General

CAUTION! Do not remove instrument protective covers. If you remove the protective instrument
panels or disable interlock devices, you may be exposed to serious hazards including, but not limited
to, severe electrical shock, laser exposure, crushing, or chemical exposure.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 77
 Appendix I Safety
I Instrument safety

Physical injury

CAUTION! Moving and Lifting Injury. The instrument is to be moved and positioned only by the
personnel or vendor specified in the applicable site preparation guide. Improper lifting can cause
painful and permanent back injury.
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 appropriate moving 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 else slides the contents out of the box.

CAUTION! Moving Parts. Moving parts can crush, pinch and cut. Keep hands clear of moving parts
while operating the instrument. Disconnect power before servicing.

Electrical safety

WARNING! Ensure appropriate electrical supply. For safe operation of the instrument:
· Plug the system into a properly grounded receptacle with adequate current capacity.
· 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 approved line cords for the
power supply in your facility.

WARNING! Disconnecting Power. To fully disconnect power either detach or unplug the power
cord, positioning the instrument such that the power cord is accessible.

78 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix I Safety
Safety and electromagnetic compatibility (EMC) standards I

Cleaning and decontamination

CAUTION! Cleaning and Decontamination. Use only the cleaning and decontamination methods
specified in the manufacturer's user documentation. It is the responsibility of the operator (or other
responsible person) to ensure the following requirements are met:

· No decontamination or cleaning agents are used that could cause a HAZARD as a result of a
reaction with parts of the equipment or with material contained in the equipment.
· The instrument is properly decontaminated a) if hazardous material is spilled onto or into the
equipment, and/or b) prior to having the instrument serviced at your facility or sending the
instrument for repair, maintenance, trade-in, disposal, or termination of a loan (decontamination
forms may be requested from customer service).
· Before using any cleaning or decontamination methods (except those recommended by the
manufacturer), users should confirm with the manufacturer that the proposed method will not
damage the equipment.

Ultraviolet (UV) Safety

The Ion Chef™ System uses a UV lamp which emits light at 254 nm. Under normal operating conditions,
the UV lamp is powered on when performing the cleaning protocol. Safety interlocks are used to ensure
that the UV lamp is not powered when the door is open.

Safety and electromagnetic compatibility (EMC) standards

The instrument design and manufacture complies with the following standards and requirements for
safety and electromagnetic compatibility.

Safety compliance
Reference Description

EU Directive 2006/95/EC European Union “Low Voltage Directive”

IEC 61010-1 Safety requirements for electrical equipment for measurement, control, and
laboratory use – Part 1: General requirements
EN 61010-1
UL 61010-1
CSA C22.2 No. 61010-1

IEC 61010-2-010 Safety requirements for electrical equipment for measurement, control and
laboratory use – Part 2-010: Particular requirements for laboratory equipment
EN 61010-2-010
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 requirements for laboratory centrifuges

IEC 61010-2-081 Safety requirements for electrical equipment for measurement, control and
laboratory use – Part 2-081: Particular requirements for automatic and semi-
EN 61010-2-081
automatic laboratory equipment for analysis and other purposes

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 79
 Appendix I Safety
I Safety and electromagnetic compatibility (EMC) standards

EMC
Reference Description

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 Electromagnetic Disturbance

Characteristics of Industrial, Scientific, and Medical (ISM) Radiofrequency
Equipment

ICES-001, Issue 3 Industrial, Scientific and Medical (ISM) Radio Frequency Generators

US FCC CFR Title 47 Part Operation within the band 13.110−14.010 MHz.
15.225, Subpart C

Industry Canada RSS 210, Licence-Exempt Radio Apparatus: Category I Equipment

Issue 8, Annex 2 EN 302
291-1/2 V1.1.1

Environmental design
Reference Description

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

Directive 2011/65/EU European Union “RoHS Directive” – Restriction of hazardous substances in

electrical and electronic equipment

80 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Appendix I Safety
Chemical safety I

Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards, ensure laboratory personnel
read and practice the general safety guidelines for chemical usage, storage, and waste provided
below. Consult the relevant SDS for specific precautions and instructions:

· Read and understand the Safety Data Sheets (SDSs) provided by the chemical manufacturer
before you store, handle, or work with any chemicals or hazardous materials. To obtain SDSs, see
the “Documentation and Support” section in this document.
· Minimize contact with chemicals. Wear appropriate personal protective equipment when handling
chemicals (for example, safety glasses, gloves, or protective 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, follow the 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 spills or leaks from the primary container.
Both containers must be compatible with the waste material and meet federal, state, and local
requirements for container 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 by the instrument is

potentially hazardous. Follow the guidelines noted in the preceding General Chemical Handling
warning.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 81
 Appendix I Safety
I Biological hazard safety

Biological hazard safety

WARNING! BIOHAZARD. Biological samples such as tissues, body fluids, infectious agents,
and blood of humans and other animals have the potential to transmit infectious diseases.
Conduct all work in properly equipped facilities with the appropriate safety equipment (for example,
physical containment devices). Safety equipment can also include items for personal protection,
such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or
goggles. Individuals should be trained according to applicable regulatory and company/ institution
requirements before working with potentially biohazardous materials. Follow all applicable local,
state/provincial, and/or national regulations. The following references provide general guidelines when
handling biological samples in laboratory environment.

· U.S. Department of Health and Human Services, Biosafety in Microbiological and Biomedical
Laboratories (BMBL), 5th Edition, HHS Publication No. (CDC) 21-1112, Revised December 2009;
found at:
https://www.cdc.gov/labs/pdf/CDC-BiosafetymicrobiologicalBiomedicalLaboratories-2009-
P.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

82 Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide
 Documentation and support

Customer and technical support

Visit thermofisher.com/support for the latest service and support information.
• Worldwide contact telephone numbers
• Product support information
– Product FAQs
– Software, patches, and updates
– Training for many applications and instruments
• Order and web support
• Product documentation
– 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 forth in the
Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/
global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at
www.thermofisher.com/support.

Ion AmpliSeq™ Library Preparation on the Ion Chef™ System User Guide 83
Ion AmpliSeq for Chef Kit_UG_MAN0013432-v29-GUID-1681BA46-5AC9-4B93-
A43A-13955A5B5213-2021/07/24 23:43:06 en
20:29:51.406+01:00
thermofisher.com/support | thermofisher.com/askaquestion
thermofisher.com

2 August 2021