Ion AmpliSeq Designer: Getting Started · reactions) for library preparation on the Ion Chef ... Ion AmpliSeq™ Designer: Getting Started User Guide 7. 1 2 3 7-11 1 Start a design

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

Ion AmpliSeq™ Designer: Getting StartedUSER GUIDEPublication Number MAN0010907

Revision G.0

Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288For 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: MAN0010907

Revision Date DescriptionG.0 18 December 2019 • Updated “Navigation bar“ on page 9 for new user interface.

• Added “Manage my account“ on page 9.

• Added Oncomine™ tumor-specific panel information and instructions: Chapter 4,“Oncomine™ tumor specific panels“.

• Added “Oncomine™ tumor specific panels FAQs“ on page 91.

• Updated “Related documentation“ on page 107.

F.0 12 June 2018 • Updated for the Ion AmpliSeq™ Designer v7.0 user interface

• Updated for Ion AmpliSeq™ HD panel design and ordering

E.0 24 May 2017 • Support added for Ion AmpliSeq™ On‑Demand Panels

• Updated for new Ion AmpliSeq™ Designer user interface

D.0 5 October 2015 • Updated screenshots for software user interface changes

• Added introduction sections for new user interface

• Support added for the Chip Calculator

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you acceptthe terms and conditions of all applicable Limited Use Label Licenses.Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registeredtrademark of Roche Molecular Systems, Inc., used under permission and license. Apple and Safari are trademarks of Apple Inc. Microsoft andInternet Explorer are trademarks or registered trademarks of Microsoft Corporation in the United States and/or other countries. Firefox is atrademark of the Mozilla Foundation. Google and Chrome are trademarks of Google LLC.

©2019 Thermo Fisher Scientific Inc. All rights reserved.

https://www.thermofisher.com/symbols-definition

Contents

■ CHAPTER 1 Get started with Ion AmpliSeq™ Designer . . . . . . . . . . . . . . . 6

Ion AmpliSeq™ Designer home page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Navigation bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Manage my account . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Chip Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Create and manage reference genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Copy existing amplicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Known limitations of the Copy Amplicons function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Basic and biological filtering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Filtering of repeat regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Biological filtering ‑ GC content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Repeat regions and RepeatMasker filtering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ CHAPTER 2 Ion AmpliSeq™ on-demand and custom panels . . . . . . . . 22

Ion AmpliSeq™ On‑Demand Panel designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Start a new Ion AmpliSeq™ On‑Demand Panel design . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Ion AmpliSeq™ custom panel designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Start a new DNA gene design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Start a new DNA hotspot design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Start a new RNA gene expression design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Start a new RNA Gene Fusion design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Browse Ion AmpliSeq™ Ready-to-Use and Community Panels . . . . . . . . . . . . . . . . . . . . . . . . 45

■ CHAPTER 3 Ion AmpliSeq™ HD custom panels . . . . . . . . . . . . . . . . . . . . . . 46

Start a new Ion AmpliSeq™ HD DNA gene design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Start a new Ion AmpliSeq™ HD DNA hotspot design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Start a new Ion AmpliSeq™ HD RNA Gene Fusion design . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

■ CHAPTER 4 Oncomine™ tumor specific panels . . . . . . . . . . . . . . . . . . . . . . 58

Oncomine™ tumor specific panel designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Workflow specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Panel design screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Price tiers for the Oncomine™ tumor specific panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Ion AmpliSeq™ Designer: Getting Started User Guide 3

Order a core Oncomine™ tumor specific panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Order a custom Oncomine™ tumor specific panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Oncomine™ tumor specific panel files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

■ APPENDIX A Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Create a BED file from a list of variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71Create a custom BED file for input into Ion AmpliSeq™ Designer . . . . . . . . . . . . . . . . . 71Start from a list of dbSNP target identifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71Use the UCSC Genome Browser to create a BED file with padded exons . . . . . . . . . . . 73

FASTA reference sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Known polymorphism BED file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Pipeline details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76The design pipeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76Tiling algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77Pooling algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77Advantages of pooling and tiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Hotspots pipeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

Ion Chip capacities for Ion AmpliSeq™ DNA libraries sequenced at equal depths . . . . . . . 79

Ion Chip capacities for Ion AmpliSeq™ RNA libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Oncomine™ tumor specific panels: a complete list of targets . . . . . . . . . . . . . . . . . . . . . . . . 80Oncomine™ Bladder Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80Oncomine™ BRCA Expanded Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Oncomine™ Colorectal and Pancreatic Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82Oncomine™ Gastric and Esophageal Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83Oncomine™ Gynecological Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84Oncomine™ Kidney Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84Oncomine™ Liver Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85Oncomine™ Lymphoma Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86Oncomine™ Melanoma Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87Oncomine™ Prostate Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Frequently asked questions (FAQs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Ion AmpliSeq™ Designer general FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89Oncomine™ tumor specific panels FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91Ion AmpliSeq™ On‑Demand Panels FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96Ion AmpliSeq™ HD Custom panels FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Related Ion sequencing products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Helpful tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

Advanced features and tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103Minimize off-target hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103Determine GC content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105Add coverageAnalysis plugin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

Contents

4 Ion AmpliSeq™ Designer: Getting Started User Guide

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Contents


Get started with Ion AmpliSeq™

Designer

■ Ion AmpliSeq™ Designer home page . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

■ Navigation bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

■ Manage my account . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

■ Chip Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

■ Create and manage reference genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

■ Copy existing amplicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

■ Basic and biological filtering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

1


Ion AmpliSeq™ Designer home page

Before you start a panel design or browse pre-designed panels, sign in at AmpliSeq.com with your Thermo Fisher Scientific user name and password. The IonAmpliSeq™ Designer home page provides tools and options for designing andordering panels. The following types of ordering options are available.

• In stock – designs that include optimized amplicons with verified performancefor germline analysis; can be customized at the gene level and configured to aspecific human disease research area; offered in small reaction packs (8 or 32reactions) for library preparation on the Ion Chef™ System and 24 or 96 reactionsfor manual library preparation.

– Ion AmpliSeq™ On-Demand panels – designs that are suitable for commondisease research areas. You can create designs by directly selecting from acatalog of > 5,000 pre-tested genes or browse the Ion AmpliSeq™ Designergene repository for specific research areas.

– Oncomine™ tumor specific panels – designs that are curated for specifictumor types with verified performance; can be ordered as-is or customized atthe gene level by selecting pre-tested genes from the Ion AmpliSeq™

Designer repository.• Made-to-order – custom designs for germline or somatic analysis of any genome

that are customizable at the amplicon level.– Ion AmpliSeq™ Custom panels – DNA and RNA designs for germline or

somatic analysis of any genome, ordered in large reaction packs.– Ion AmpliSeq™ HD Custom panels – ultra-high sensitivity DNA and RNA

designs for germline or somatic analysis of the human genome; designed forsequencing workflows with Ion AmpliSeq™ HD chemistry that features dualbarcoded amplicons and bidirectional sequencing.

• Fixed Panels – RNA and DNA panels with a fixed design for germline or somaticanalysis, ordered in small reaction packs.

– Community Panels – fixed-design DNA and RNA panels for germline orsomatic analysis that are created by a 3rd party and promoted by ThermoFisher Scientific.

– Ready-to-Use Panels – in-stock fixed-design DNA and RNA panels forgermline or somatic analysis that are created by Thermo Fisher Scientificresearchers.

Chapter 1 Get started with Ion AmpliSeq™ DesignerIon AmpliSeq™ Designer home page 1


https://www.ampliseq.com

1 2 3

7-11

1 Start a design using In stock panels and genes2 Order Fixed Panels3 Create custom Made-to-order panels

Chapter 1 Get started with Ion AmpliSeq™ DesignerIon AmpliSeq™ Designer home page1


Navigation bar

Use the navigation bar to access the main tools in Ion AmpliSeq™ Designer.

1 2 3 4 5 6 7

8 9 10

1 Home—The home page is the starting point for creating a panel design.2 My Designs—Browse the panel designs that you have already created.3 Notifications—View updates on new features and messages from the Ion AmpliSeq™ Designer.4 Chip Calculator—Calculate the number of sample libraries that you can load per chip given your panel, sample source,

and chip type. For more information, see “Chip Calculator“ on page 10.5 Genomes—View a list of public genome references that are available in Ion AmpliSeq™ Designer, or create a custom

reference. For more information, see “Create and manage reference genomes“ on page 11.6 Fixed Panels—Browse the ready-to-use and community panel designs.7 Help—Open the Ion AmpliSeq™ Designer Help in a separate tab in your browser.8 Search—Search for available panels by gene name, symbol, or keyword.9 Live Chat—Chat with an Ion AmpliSeq™ Designer expert to get help and advice.

10 My Account—View and manage your Thermo Fisher Scientific account, profile information, orders, shopping carts, andfavorites on thermofisher.com. For more information, see “Manage my account“ on page 9.

Manage my account

You can manage your Ion AmpliSeq™ Designer account from any screen. Click MyAccount then make one of the following selections from the dropdown list. Eachselection redirects you to an appropriate page on thermofisher.com.

Selection Description

Account Access your orders, profile, online services, and tools or upgrade youraccount.

Orders View and manage recent orders.

Shopping Cart View and manage saved carts or add a new cart.

Favorites View and manage a list of frequently-ordered products or create a newlist.

Profile Manage your contact information, passwords, and security.

Sign Out Sign out of Ion AmpliSeq™ Designer.

Chapter 1 Get started with Ion AmpliSeq™ DesignerNavigation bar 1


http://www.thermofisher.com


Chip Calculator

The Chip Calculator can be accessed through the navigation bar. Use the ChipCalculator to view recommendations for maximum sample library loading per chipfor a given panel, chip, and coverage (2,000X for somatic samples, and 30X forgermline samples). You can use the Chip Calculator as a guide for library loading forReady-to-Use panels, and to aid in the design of a Made-to-Order panel.

The Chip Calculator is also available by clicking the Review panel button in theActions column of a Ready-to-Use panel, and in the Results Ready screen that isreturned after you submit a Made-to-order panel design.

As you proceed down the information list, the options become application-specific.Also note that instrument selection changes the chip options in the lower table.

The panel-specific chip calculator contains pre-populated fields pertaining to thatpanel.

Chapter 1 Get started with Ion AmpliSeq™ DesignerChip Calculator1


For information on chip capacity, see “Ion Chip capacities for Ion AmpliSeq™ DNAlibraries sequenced at equal depths“ on page 79 and “Ion Chip capacities for IonAmpliSeq™ RNA libraries“ on page 80.

Create and manage reference genomes

In Ion AmpliSeq™ Designer, you can use various human, animal, and plant referencegenomes to build your panels. You can also upload a custom reference.

1. In the navigation bar, click Genomes.In the Design References screen, public genomes are displayed. If you havepreviously uploaded custom references, click the Custom References tab to viewthem.

Chapter 1 Get started with Ion AmpliSeq™ DesignerCreate and manage reference genomes 1


Public genomes are the available choices when you start a new design. The list ofpublic genomes in this screen is for informational purposes. You cannot select apublic genome at this point.

2. To upload a new custom reference, click Add reference.

Chapter 1 Get started with Ion AmpliSeq™ DesignerCreate and manage reference genomes1


3. Complete the required information in the Add a custom reference screen.

1

2

3

4

5

6

7

1 Reference Name—Use US-ASCII letters, numbers, and spaces, between 3 and 32characters in length.



2 Associated organism for primer specificity check—Click to view the list of organisms.If your data are associated with a supported organism, providing this information canimprove primer specificity to your custom reference by favoring primers with fewoptimal binding sites in the consensus sequence. Primer specificity check refers tothe process of identifying potential primer mispriming events. Primers with highnumber of potential mispriming events are avoided in panel designs.

3 Reference source (Recommended)—The name of the database/source of the DNAsequence.

4 Reference description—Add any notes regarding the custom reference sequence.5 Reference sequences—You can upload a FASTA file (the default size is 2.0 GB;

however, on request the limit can be extended to 4.0 GB), or you can copy and pastethe reference sequence.

6 Genome short name for Torrent Server—Use lowercase US-ASCII letters, numbers,and underscores, between 1 and 30 characters in length.

7 Known polymorphism (BED) file—Indicates regions of the sequences in the customreference FASTA file with high polymorphism (that is, SNPs, INDELs, or othervariation). Ion AmpliSeq™ Designer minimizes primer overlap with these regions. Thisfile is optional. For specifications on creating and formatting BED files for uploading,see “Known polymorphism BED file“ on page 75.

4. Under Reference sequences (FASTA file) click Select file, browse to the locationof the FASTA file on your drive, then select it.

5. If appropriate, under Known polymorphism (BED) file, click Select file toupload the polymorphism BED file.

6. After the upload progress bars complete, click Save.

7. When the upload procedure finishes, click the Custom References tab to viewyour uploaded reference.



8. Click the custom reference name to show more information.



9. Click Edit at the upper right to edit the reference name, reference source, andreference description.

Note:· Updates to these textual identifiers are made throughout the entire site.· You cannot change the uploaded files (genomic data) as they are permanently

associated with this assigned custom reference genome. To make changes,delete and re-upload your edited files using the Add reference button. ClickDelete to remove the reference from the list of active custom references. Thisaction does not affect existing designs — associated custom references are stilldownloadable.



10. When building your custom panel, click the Custom Reference tab, then selectyour custom reference from the Custom Reference list.

Copy existing amplicons

In Ion AmpliSeq™ Designer, you can copy amplicons from one or more IonAmpliSeq™ ready-to use panels, community panels, and previous made-to-orderdesigns, then resubmit into the pipeline to generate new designs, without needing tocalculate the position of the amplicon.

1. Create a new draft design from the home screen, determine the source of theamplicons you want to copy from (ready-to-use panels, community panels,and/or previous designs), then copy amplicons from ready-to-use and/orcommunity panels.

a. In the Ready-to-Use Panels tab, click the Browse by DNA or RNA button,or in the Made-to-Order Panels tab, click Pre-designed by our Community.

b. Select the panel that you want to subset from, then click Review panel.

Chapter 1 Get started with Ion AmpliSeq™ DesignerCopy existing amplicons 1


2. In the row of your gene of interest, select the checkbox to select the gene, thenclick Copy Amplicons to copy the selected amplicons to your new design.

3. In the Copy Amplicons dialog box, select either Copy checked target rows, orCopy unchecked targets instead of checked target rows, then click Copyamplicons to another design.

• You can also click Download amplicon list to download a CSV file of thecomplete amplicon list to edit the file and upload it to a custom design.

• You can copy the entire amplicon list from a panel by clicking the CopyAmplicons button for the panel in the panel list page, when the button isactive.

4. Complete the Select Destination dialog box.

• Select a preexisting design from the Destination list as a destination for thecopied amplicons, or

• Select New design..., then in New design name, enter a name.

Click Copy amplicons to copy the amplicons to the selected design.

Chapter 1 Get started with Ion AmpliSeq™ DesignerCopy existing amplicons1


If you are copying amplicons into a preexisting design, amplicons must be of thesame genome, or in draft form.

5. After you compile a list of targets for which you want to create a new design,click Submit targets, then confirm your submission.For information on how to submit made-to-order designs, see “Start a new DNAgene design“ on page 28.Subset designs can be combinations of predesigned regions and amplicons fromcustom designs mixed and matched with amplicons from other custom designsor ready-to-use and community panels.

• Custom amplicons from a private design that is shared with you by a colleaguecannot be copied using the Add Amplicon By ID form or the Upload Filemethod. Amplicons can only be copied using the Copy Amplicons button anddialog boxes in the user interface.

• The following DNA panels are not compatible for use with the Copy Ampliconsfunction.

– HID—Precision ID Identity Panel (Cat. No. A25643)– HID—Precision ID Ancestry Panel (Cat. No. A25642)– Ion AmpliSeq™ Exome RDY Kit (Cat. No. A38262)– Ion AmpliSeq™ Colon and Lung Cancer Panel– Ion AmpliSeq™ On-Demand panels– Oncomine™ tumor specific panels

• RNA panels are also not compatible for use with the Copy Amplicons function,because RNA designs are based on predesigned primers for every transcript inour database. For RNA designs, use the Copy Targets function to create Made-to-Order RNA designs containing the same amplicons.

• Amplicons that are greater than 200 bp in size cannot be copied to Ion AmpliSeq™

HD designs.

Known limitationsof the CopyAmpliconsfunction

Chapter 1 Get started with Ion AmpliSeq™ DesignerCopy existing amplicons 1


Basic and biological filtering

The human genome is heavily populated with repeat regions that make designingprimers difficult, a well known challenge in polymerase chain reaction (PCR) design.Ion AmpliSeq™ Designer has been developed to deliver the most robust set ofamplicons it can generate. The software specifically excludes amplicons that areplaced in repeat elements or other hypervariable regions to generate the best possibleoutcome for actual amplicon coverage when used in a reaction.

A focus of the Thermo Fisher Scientific Research & Development department is tobetter understand the properties of repeat regions to allow primer placement in theseregions to achieve higher target design rate while maintaining coverage uniformityand on-target rates.

The biological filtering mechanism that is incorporated into the Ion AmpliSeq™

Designer pipeline to evaluate repeat elements is the RepeatMasker program.RepeatMasker is a program that screens DNA sequences for interspersed repeats andlow complexity DNA sequences. The output of the program is a detailed annotationof the repeats that are present in the query sequence as well as a modified version ofthe query sequence in which all the annotated repeats have been masked (default:replaced by Ns). The RepeatMasker program is an annotation track that is availablethrough the UCSC Genome Browser. Ion AmpliSeq™ Designer links directly to thebrowser and offers users the visual representation to distinguish between three BEDfiles as custom annotation tracks.

• The resulting BED file for the design that was submitted (the data appears underthe "InputTargets" blue label in the UCSC browser)

• The resulting BED file for the design that is generated by the application (the dataappears under the "CoveredBases" green label in the UCSC browser)

• The difference between these two BED files (the data appears under the"MissedBases" red label in the UCSC Genome Browser)

While G-C bonds contribute more to the stability, and therefore increased meltingtemperature, of primer/template binding than do A−T bonds, it is important to notethat two primer/template complexes with similar or even identical GC content canresult in a different melting temperature because of base order influence on overallstability.

GC-rich regions for the target DNA are difficult to amplify, and are generally avoidedwhen defining an in silico algorithm.

When submitted as a manual target region with coordinates chr13:48877883-49056026,the resulting coverage was 38.31% with 4 pools and 611 amplicons.

Filtering of repeatregions

Biologicalfiltering ‑ GCcontent

Repeat regionsandRepeatMaskerfiltering

Chapter 1 Get started with Ion AmpliSeq™ DesignerBasic and biological filtering1


In closer examination of the design results, it is apparent that the majority of thisregion is interspersed with repeat elements.

An attempt to redesign the region by input type Gene + UTR results in coverage of92.58% with 4 pools and 60 amplicons.

Chapter 1 Get started with Ion AmpliSeq™ DesignerBasic and biological filtering 1


Ion AmpliSeq™ on-demand andcustom panels

■ Ion AmpliSeq™ On-Demand Panel designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■ Ion AmpliSeq™ custom panel designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

■ Browse Ion AmpliSeq™ Ready-to-Use and Community Panels . . . . . . . . . . . . . 45

Ion AmpliSeq™ On‑Demand Panel designs

Ion AmpliSeq™ On-Demand panel designs are suitable for common disease researchareas. They are customizable at the gene level.

Up to 800 genes are available in Ion AmpliSeq™ Designer for design creation, and upto 500 genes are allowed for order.

Ion AmpliSeq™ on-demand panels bring smaller pack sizes and a performance-guaranteed gene catalog to customized next-generation sequencing panels. Sequenceyour genomic targets using as little as 10 ng of input DNA. Simple, fast, andaffordable targeted sequencing of specific genes or genomic regions using the IonPGM™ System is accessible for any lab conducting human disease research.

These on-demand panel designs are previously tested, readily available, and shipwithin a few days.

Ion AmpliSeq™ On-Demand Panels represent an alternative way to design panels foruse in inherited disease (germline mutation) research applications. Ion AmpliSeq™

On-Demand Panels can be designed by selecting genes using a content selectionengine, or uploading your own gene list. Each panel consists of two DNA primerpools, and the number of primer pairs per pool depends on the genes you select. IonAmpliSeq™ On-Demand Panels are limited to 500 genes and/or 15,000 amplicons. Thegene designs in the On-Demand catalog have been optimized for high performance.You can add genes that are not in the On-Demand catalog to your panel as a Spike-inpanel. Spike-in panels are created as 2-pool designs and are limited to 123 ampliconsper pool.

2

Start a new IonAmpliSeq™

On‑Demand Paneldesign


This section covers how to design an Ion AmpliSeq™ On-Demand Panel using thefeatures and tools available in Ion AmpliSeq™ Designer.

1. On the home page, in the Ion AmpliSeq™ On-Demand Panels section, click Orbrowse disease research areas.

2. On the Browse Disease Research Areas screen, scroll to an area of interest, thenclick > to expand the area and view subcategories to narrow your focus.

The number in parentheses is the number of genes that are included in thedisease category.

Chapter 2 Ion AmpliSeq™ on-demand and custom panelsIon AmpliSeq™ On‑Demand Panel designs 2


3. When you have found a Disease Research Area that most closely matches yourinterest, select the category by clicking its checkbox.

Note:· You can select Disease Research Areas from different categories to have them

display additively in the Selections pane.· The total number of on-demand genes in your final panel order cannot exceed

500, and the total number of amplicons cannot exceed 15,000. If the totalnumber of genes or amplicons in your selections exceeds these limits, youneed to deselect genes in the design step to bring the totals within the limits.

· You can also use the search box in the upper right corner to add DiseaseResearch Areas to your design.

4. When you have completed your selections, enter a design name, then clickProceed.

5. On the panel order page, you can perform the following functions.• Add additional genes to the panel design, either individually or as an

uploaded list.• Remove a gene from the design by unchecking its checkbox.• Use the Integrative Genomics Viewer (IGV) to view the exon structure,

amplicon coverage, missed regions, and general information for each gene.• Create a Spike-in Panel by selecting Disease Research Area genes.• Order a panel.• Copy or clone an ordered and locked panel to make further changes.

Chapter 2 Ion AmpliSeq™ on-demand and custom panelsIon AmpliSeq™ On‑Demand Panel designs2


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1 Order—click when you have finished your panel design.2 Clone Panel—copy the panel and add or delete genes.3 Download Results—download results files after a design has been ordered, or a Spike-in Panel has been

created/ordered.4 Export Targets—generate an Excel CSV file of the target list.5 Upload File—upload a .csv file containing a list of genes to include in a panel.6 Add Gene—enter gene symbol to upload genes to panel design individually.7 Grid/Table—toggle between the grid (shown) and table views of target genes.8 Sort By—sort target list alphabetically or by score (a ranking of the relationship for researching the correlation between

the disease and gene).9 Genes available as on-demand or not available as on-demand (Spike-in Panel).

In the Grid view, click a gene to view its genomic and amplicon information inthe IGV.

Chapter 2 Ion AmpliSeq™ on-demand and custom panelsIon AmpliSeq™ On‑Demand Panel designs 2


6. To add genes not available as on-demand, select them, then click Create togenerate a Spike-in Panel.

Chapter 2 Ion AmpliSeq™ on-demand and custom panelsIon AmpliSeq™ On‑Demand Panel designs2


7. Click Submit in the dialog box that appears to submit the Spike-in Panel design.

IMPORTANT!· Spike-in panel designs are limited to 123 amplicons per pool to be compatible

with the spike-in process.· Your On-Demand design is locked after submitting a Spike-in design, and can

be edited only if it is cloned.

8. When you have completed your design, click Order.Your order should arrive in a few days.

Ion AmpliSeq™ custom panel designs

If the Ion AmpliSeq™ ready-to-use panels don't match your gene targets, then simplyselect a subset of the targets or enter your specific genomic content online to designand create customized Ion AmpliSeq™ next-generation sequencing gene panels justfor you.

Custom panel designs are customizable for any genome at the amplicon level. Panelscan be designed against a number of preloaded reference genomes, or for any otherorganism by uploading your own FASTA file reference sequence.

Custom panels are delivered with a few weeks in tubes of prepooled, multiplexedprimers in ready-to-use concentrations. You can add 384-well plates to your order, inwhich each well contains individual primer pairs in concentrated format. For custompanels with ≤96 primer pairs, you will receive 2,250 total reactions. For custom panelswith >96 primer pairs, you will receive 9,000 total reactions.

Ion AmpliSeq Made-to-Order panels, when ordered in the Tubes Only format, contain750 reactions per pool for panels with ≤96 amplicons, or 3,000 reactions per pool forpanels with >96 amplicons. Panels ordered in the Tubes Plus 384-Well Plate formatcontain 2,250 reactions (750 in pools and 1,500 in plate format) for panels with ≤96

Chapter 2 Ion AmpliSeq™ on-demand and custom panelsIon AmpliSeq™ custom panel designs 2


amplicons, or 9,000 reactions (3,000 in pools and 6,000 in plate format) for panels with>96 amplicons.

If you are using one of the Ion AmpliSeq™ standard genome references, you candesign a custom DNA gene panel to discover new variants and detect known SNPsand INDELs in your genes of interest.

1. On the home page, in the Ion AmpliSeq™ Custom panels section, click Addcustom targets.

2. In the Start a new AmpliSeq design screen, in the Name and details step, entera design name and optional details.

3. In the Application Type step, click DNA Gene designs (multi-pool).Clicking the application type filters the compatible genomes that are shown.

4. In the DNA Type step, select a DNA type.

5. In the Select genome to use step, select a genome to use as reference. If you havean Ion AmpliSeq™ custom references, click Custom Reference.Custom genome references are compatible only with Ion AmpliSeq™ DNAdesigns.

6. Click Next: Add Targets.

Start a new DNAgene design

Chapter 2 Ion AmpliSeq™ on-demand and custom panelsIon AmpliSeq™ custom panel designs2


7. Add targets using the available options. For information and examples abouthow to add the targets, click Input Specifications.

Option Description

Add Gene/Region Add targets manually.

1. Select type: Gene (CDS only), Gene (CDS +UTR), or Region.

2. Start typing the gene symbol or region, then select the genefrom the list.

3. Click Add target after each entry. A green or red text boxappears after each target to let you know if the target wasadded successfully.

4. When finished, click Submit targets.

Add Amplicon byID

Enter the amplicon IDs assigned to specific genomiccoordinates.

Upload File Upload genomic coordinates of several targets at one time usinga CSV or BED file.

1. Click Choose File, browse to a file that contains fusiontargets, then select the file.

2. Click Upload.

The software uploads the targets, checks them, and verifies regions.



8. For any highlighted erroneous target, either correct the coordinates inside thetable, or select the checkbox, then click Delete to remove that target.

9. Select an Exon padding value if different from the standard setting.

10. Select a DNA Type for the type of sample to use to construct the library:Standard DNA, FFPE DNA, or cfDNA.This setting affects amplicon length in the panel design.

11. (Optional) Click Export targets to download your targets into a CSV file.Open the file in Microsoft™ WordPad or Microsoft™ Excel™.

12. To submit your panel, click Submit targets.Up to 5 designs can be submitted at one time.

13. Click OK to confirm your submission.When confirmed, you see a confirmation message and receive an emailconfirming the design submission.

14. When the assay design results are ready, you receive an email notificationinstructing you to review the results in Ion AmpliSeq™ Designer. Click the Viewresults link provided in the email to be directed to the results page (or navigateto AmpliSeq.com, then click the notification or navigate to the completed designusing the My Designs tab).




15. Review your order.

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1 Switch design—Click to change the design in view.2 Edit—Edit Design Name and Details.3 Copy Targets—Copy your design to modify it.4 Start a new design—Starts a new design.5 Add to cart—When this button is green, click it to add the highlighted design to your cart. If there are not sufficient

amplicons (at least 12 per pool), the button is unavailable and a message appears.6 Chip Calculator—Obtain Use the Chip Calculator to obtain guidelines for sample library loading with various Ion chips to

help in panel design.7 Download results—Design data results are available for download when your assay design is complete. A compressed

folder downloads containing several results files.8 Sharing—Creates a link to your designs that you can email to another Ion AmpliSeq™ Designer account holder.

Sharing your design also makes your custom reference available to review and download by anyone to whom you providethe link to the design.

9 Export targets—Downloads your targets into a CSV file.10 Copy amplicons—Allows you to copy your amplicons and download an amplicon list, or copy your amplicons to another

design.11 View Cart—After you add your designs to your cart, you can view the cart and request a quote.



You can design a custom DNA hotspot panel to detect known SNPs and INDELs inyour genes of interest.



3. In the Application Type step, click DNA Hotspot designs (single-pool).Clicking the application type filters the compatible genomes that are shown.

4. In the DNA Type step, select a DNA type.

5. In the Select genome to use step, select a genome to use as reference. If you havean Ion AmpliSeq™ custom reference, click Custom Reference.Custom genome references are compatible only with Ion AmpliSeq™ DNAdesigns.


Start a new DNAhotspot design



7. Add hotspot targets using the available options. For information and examplesabout how to add the hotspot targets, click Input Specifications.

Option Description

Hotspot by ID Add targets manually

1. Enter the SNP ID, or start typing and select the full ID fromthe list.

2. Click Add target after each entry. A green or red text boxappears after each to let you know if the target was addedsuccessfully.


Hotspot byCoordinates

Enter hotspot IDs assigned to specific genomic coordinates.

Upload File Upload genomic coordinates of several targets at one time usinga SNP list or BED file.


2. Click Upload.

The software uploads the targets, checks them, and verifies regions.

8. For any highlighted erroneous target, either correct the coordinates inside thetable, or select the checkbox, then click Delete to remove that target.

9. Select a DNA Type for the type of sample that you plan to construct the libraryfrom: Standard DNA, FFPE DNA, or cfDNA.This setting affects amplicon length in the panel design.











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You can design a custom RNA gene expression panel to measure differentialexpression of genes of interest in your sample types.



3. In the Application Type step, click RNA Gene Expression designs (single-pool).Clicking the application type filters the compatible genomes shown.

4. In the Select genome to use step, accept the default Human (RefSeq).Human (RefSeq/Ensembl) is the only reference sequence that is allowed withthis panel type.


Start a new RNAgene expressiondesign



6. Add gene targets using the available options. For information and examplesabout how to add the targets, click Input Specifications.

Option Description

AddGene/Transcript

Add targets manually.

1. Start typing the gene symbol or RefSeq accessionnumber, then select from the list

2. Click Add target after each entry. A green or red text boxappears after each to indicate if the target was addedsuccessfully.


Upload File Upload many targets at one time using a CSV or text file.


2. Click Upload.

The software uploads the targets, and checks them.

7. For any highlighted erroneous target, either correct the coordinates inside thetable, or select the checkbox, then click Delete to remove the target.








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Select the RNA Gene Fusion design option to make an RNA Gene Fusion panel todetect expression of known gene fusions of interest in your samples.

IMPORTANT! For successful panels, you must have at least 12 gene expression (GEX)assays per panel. We provide 12 default GEX assays for each panel that you can acceptor replace with targets of your choice. For panels requiring two pools, the GEX assaysare split between the two pools.




4. In the Application Type step, click RNA Gene Fusion Designs (multi-pool).

5. In the Select genome to use step, accept the default Human (RefSeq).Human (RefSeq/Ensembl) is the only reference sequence that is allowed withthis panel type.

6. Click Next: Add Targets.A screen appears for adding gene fusion targets to the panel.

Start a new RNAGene Fusiondesign



7. Add fusion targets using the available options. For information and examplesabout how to add targets, click Input Specifications.

Tab Description

Add Fusion Add targets manually.

1. Start typing the gene symbol of the gene that you want to addtargets for, then select your targets from the list of available genefusions.



1. Click Choose File, browse to a file that contains fusion targets,then select the file.

2. Click Upload.

After you select a fusion symbol pair, available isoforms for the pair appear in atable under the Symbol Pair field.



8. Add the fusions by selecting the checkbox in the row of each target, then clickAdd fusion(s).

The software uploads the targets and checks them. After completion, a statusmessage appears at the top of the screen: either "Target saved successfully" ingreen text, or "# duplicate fusions ignored" in red text.

9. Repeat step 7 and step 8 to add additional fusion targets if needed.As you add fusion targets, the targets appear in a panel table at the bottom of thescreen.

Proceed to “Add gene expression assays“ on page 42 to add or edit gene expressionassays and finish the design.



Add gene expression assays

Every fusion panel is required to have 12 gene expression assays, and 12 arepreselected by default to get you started. To pick your own gene expression assays,delete the default assays and add your own gene expression targets by gene symbolor RefSeq transcript accession.

1. Click the Add Genes/Transcript tab.

2. At the top of the screen, enter valid gene symbols (preferably an HGNC-approved symbol) as in the following example, or valid RNA RefSeq accessionnumbers, then click Add target.

Alternatively, you can upload your own gene expression assays. Click InputSpecifications for more details and a CSV template for creating your own list ofgenes or RefSeq Accession numbers.If you want to restore the prepopulated gene expression assays, click Restorepre-populated genes.

3. Repeat step 2 to add additional genes or RNA RefSeq accession numbers.

4. To submit your design after you have selected your gene fusion targets and geneexpression assays, click Submit targets.5 designs can be submitted at a time.

5. Confirm your submission.You can submit up to 4 more designs, if needed.Acknowledgement of the submission appears at the top of the page. An emailnotification is sent when the results are ready for review.

6. When your fusion results are ready, you receive an email notification instructingyou to review the results in Ion AmpliSeq™ Designer.You may need to check your spam folder and move the email to your Inbox toenable its links.



7. Click the link provided in the email notification, or go to the Ion AmpliSeq™

Designer website and navigate to My Designs4RNA.

8. In the Design column, click the name of your design.

9. Review results in the Fusions tab, then make changes if needed by clicking CopyTargets and submitting a new design with your changes.

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1 Switch design—Click to change the design in view.2 Edit—Edit the design name and details.3 Copy Targets—Copy your design to modify it.4 Add design—Start a new design. After you have reviewed your designs, you can place the order.5 Add to cart—When this button is green you can click it to add the highlighted design to your cart.6 Download results—Design data results are available for download after your assay design is complete. A compressed

folder downloads containing various results files.7 Export targets—Downloads your targets as a CSV file.8 Copy Amplicons—Not applicable for RNA designs.9 View Cart —After you add your designs to your cart you can view the cart and request a quote.

10. In the Genes tab, review gene assay results.



11. Click the Gene link to view compatible transcripts.



Browse Ion AmpliSeq™ Ready-to-Use and Community Panels

1. In the home page, in the navigation bar, click Fixed Panels, then clickCommunity Panels or Ready-to-Use Panels in the dropdown list.The Ion Research Panels screen opens, which displays a list of availableCommunity Panels or Ready-to-Use Panels, their descriptions, and available useractions.

2. To filter the list of panels, in the left filter pane, under Research Area, select oneor more areas of interest, such as Cancer Research. Alternatively, in the Searchfield, enter a key word, then click Go or press Return to search for a specificpanel.

Basic panel information is presented in the table, but you can view more detailsby clicking More and Review Panel. Click Download panel files to obtain panelfiles required for analysis of your sequencing results.

Chapter 2 Ion AmpliSeq™ on-demand and custom panelsBrowse Ion AmpliSeq™ Ready-to-Use and Community Panels 2


Ion AmpliSeq™ HD custom panels

■ Start a new Ion AmpliSeq™ HD DNA gene design . . . . . . . . . . . . . . . . . . . . . . . . 46

■ Start a new Ion AmpliSeq™ HD DNA hotspot design . . . . . . . . . . . . . . . . . . . . . 50

■ Start a new Ion AmpliSeq™ HD RNA Gene Fusion design . . . . . . . . . . . . . . . . . 54

Ion AmpliSeq™ HD custom panel designs are customizable for any genome at theamplicon level, with ultra-high sensitivity for various research applications.

Ion AmpliSeq™ HD technology is a new amplicon-based library preparationtechnology for Ion Torrent™ next-generation sequencing that gives you the power todesign your own panels and find variants with a low limit of detection-down to 0.1%for cell-free DNA samples. This revolutionary technology enables researchers toroutinely get answers when ultra-high sensitivity is required, such as when detectinglow-frequency alleles in circulating tumor DNA or trace pathogenic microbial speciesin blood.

These made-to-order custom panel designs ship within a few weeks.

Ion AmpliSeq Made-to-Order panels, when ordered in the Tubes Only format, contain750 reactions per pool for panels with ≤96 amplicons, or 3,000 reactions per pool forpanels with >96 amplicons. Panels ordered in the Tubes Plus 384-Well Plate formatcontain 2,250 reactions (750 in pools and 1,500 in plate format) for panels with ≤96amplicons, or 9,000 reactions (3,000 in pools and 6,000 in plate format) for panels with>96 amplicons.

Start a new Ion AmpliSeq™ HD DNA gene design

Ion AmpliSeq™ HD panels provide ultra-high sensitivity for your targeted sequencingapplications. Select the DNA Gene design option to design a panel to discover newvariants and detect known SNPs and INDELs in your genes of interest.

1. In the home page, in the Ion AmpliSeq™ HD Custom panels section, click Addcustom targets.

2. In the Start a new AmpliSeq HD design screen, in the Name and details step,enter a design name and optional details.

3. Select your Application type: DNA Gene designs (multi-pool).

4. In the Select genome to use step, accept the default.Human hg19 genome reference is the only reference sequence that is allowedwith this panel type.


3



Option Description

Add Gene/Region Add targets manually.

1. Select type: Gene (CDS only), Gene (CDS +UTR), or Region.

2. Start typing the gene symbol or region, then select thegene from the list.



Add Amplicon byID

Enter amplicon IDs assigned to specific genomic coordinates.

Upload File Upload genomic coordinates of several targets at one timeusing a CSV or BED file (select from the "Type" list).

Ion AmpliSeq™ HD panel design numbers (shown at upper left) are designatedwith an "IAH" prefix. Standard Ion AmpliSeq™ panel designs are designatedwith an "IAD" prefix.Ion AmpliSeq™ Designer uploads the targets, checks them, and verifies regions.Ion AmpliSeq™ HD panel designs are limited to 5,000 amplicons per primer pool.Contact Thermo Fisher Scientific regarding Ion AmpliSeq™ HD panels thatrequire more than two primer pools.

7. For any highlighted erroneous target, either correct the coordinates inside thetable, or remove them by checking their checkbox, then clicking Delete.

8. Select an Exon padding value if different from the standard setting.

Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD DNA gene design 3


9. Select a DNA Type for the type of sample you plan to construct the library from,either FFPE DNA, or cfDNA.This setting affects amplicon length in the panel design.


11. To submit your panel, click Submit targets, then click OK to confirm yoursubmission.Up to 5 designs can be submitted at one time.When confirmed, you see a confirmation message and receive an emailnotification that confirms the design submission.

12. When the assay design results are ready, you receive an email notificationinstructing you to review the results in Ion AmpliSeq™ Designer. Click the Viewresults link that is provided in the email notification to be directed to the resultspage (or navigate to AmpliSeq.com, then click the notification or navigate to thecompleted design using the My Designs tab).

Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD DNA gene design3



13. Review your order, then order your panel.

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1 Switch design—Click the list to change the design in view.2 Edit—Edit the design name and details.3 Copy Targets—Copy your design to modify it.4 Start a new design—Start a new design.5 Chip Calculator—Use to obtain guidelines for sample library loading with various Ion chips to help in panel design.6 Add to cart—When this button is green you can add the highlighted design to your cart.

If there are not sufficient amplicons (at least 12), the button is unavailable and a message appears.7 Download results—Design data results are available for download when your assay design is complete. A compressed


Sharing your design makes your custom reference available for review and downloading by anyone to whom you providethe link to the design.

9 Export targets—Download your targets into a CSV file.10 Copy amplicons—Copy your amplicons and download an amplicon list, or copy your amplicons to another design.11 View Cart—After you add your designs to your cart, view the cart and request a quote.

Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD DNA gene design 3


Start a new Ion AmpliSeq™ HD DNA hotspot design

Select the DNA Hotspot design option to design a panel to detect known SNPs andINDELs in your genes of interest.

1. In the home page, in the Ion AmpliSeq™ HD Custom panels section, click Addcustom targets.

2. In the Start a new AmpliSeq HD design screen, in the Name and details step,enter a design name and optional details.

3. Select DNA Hotspot designs (single-pool).

4. In the Select genome to use step, accept the default.Human hg19 genome reference is the only reference sequence that is allowedwith this panel type.


Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD DNA hotspot design3



Option Description

Hotspot by ID Add targets manually.

1. Enter the SNP ID, or start typing and select the full ID fromthe list.



Hotspot byCoordinates

Enter hotspot IDs assigned to specific genomic coordinates.

Upload File Upload genomic coordinates of several targets at one time usingan SNP list or BED file (select from the "Type" list).

Note: Ion AmpliSeq™ HD panel design numbers (shown at upper left) aredesignated with an "IAH" prefix. Standard Ion AmpliSeq™ panel designs aredesignated with an "IAD" prefix.

Ion AmpliSeq™ Designer uploads the targets, checks them, and verifies regions.Ion AmpliSeq™ HD panel designs are limited to 5,000 amplicons per primer pool.

7. For any highlighted erroneous targets, either correct the coordinates inside thetable, or remove them by checking their checkboxes, then clicking Delete.

8. Select a DNA Type for the type of sample you plan to construct the library from:FFPE DNA, or cfDNA.This setting affects amplicon length in the panel design.

Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD DNA hotspot design 3



10. To submit your panel, click Submit targets, then click OK to confirm yoursubmission.Up to 5 designs can be submitted at one time.When confirmed, you see a confirmation message and receive an emailnotification that confirms the design submission.

11. When the assay design results are ready, you receive an email notificationinstructing you to review the results in Ion AmpliSeq™ Designer. Click Viewresults in the email notification to be directed to the results page (or navigate to AmpliSeq.com, then click the notification or navigate to the completed designusing the My Designs tab).

Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD DNA hotspot design3



12. Review your order, then order your panel.

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1 Switch design—Click the list to change the design in view.2 Edit—Edit the design name and details.3 Copy Targets—Copy your design to modify it.4 Start a new design—Start a new design.5 Chip Calculator— Use to obtain guidelines for sample library loading with various Ion chips to help in panel design.6 Add to cart—When this button is green you can add the highlighted design to your cart.





Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD DNA hotspot design 3


Start a new Ion AmpliSeq™ HD RNA Gene Fusion design

Select the RNA Gene Fusion design option to make an RNA Gene Fusion panel todetect expression of known gene fusions of interest in your samples.

IMPORTANT! We provide 19 GEX assays for each Ion AmpliSeq™ HD RNA GeneFusion panel that are fixed and uneditable.

1. On the home page, in the Ion AmpliSeq™ HD Custom panels section, click Addcustom targets.

2. In the Start a new Ion AmpliSeq HD design screen, in the Name and detailsstep, enter a design name and optional details.

3. Select RNA Gene Fusion designs (single-pool).

4. In the Select genome to use step, accept the default.Human (RefSeq/Ensembl) is the only reference sequence that is allowed with thispanel type.

5. Click Next: Add Targets.A screen appears for adding gene fusion targets to the panel.

Note:· Ion AmpliSeq™ HD panel design numbers (shown at upper left) are

designated with an "IAH" prefix. Standard Ion AmpliSeq™ panel designs aredesignated with an "IAD" prefix.

· Gene expression controls for Ion AmpliSeq™ HD are fixed and not editable,unlike Ion AmpliSeq™ Made-to-Order fusion designs.

Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD RNA Gene Fusion design3


6. Add fusion targets using the available options. For information and examplesabout how to add targets, click Input Specifications.

Tab Description

Add Fusion Add targets manually.

1. Start typing the gene symbol of the gene that you want to addtargets for, then select your targets from the list of available genefusions.



1. Click Choose File, browse to a file that contains fusion targets,then select the file.

2. Click Upload.

After you select a fusion symbol pair, available isoforms for the pair appear in atable under the Symbol Pair field.

Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD RNA Gene Fusion design 3


7. Add the fusions by selecting the checkbox in the row of each target, then clickAdd fusion(s).

The software uploads the targets and checks them. After completion, a statusmessage appears at the top of the screen indicating either that the targets weresave successfully in green text, or the number of duplicate fusions that wereignored in red text.

8. Repeat step 6 and step 7 to add additional fusion targets as needed.As you add fusion targets, the targets appear in a panel table at the bottom of thescreen.


10. To submit your panel, click Submit targets, then click OK to confirm yoursubmission.Up to 5 designs can be submitted at one time.When confirmed, you see a confirmation message and receive an emailnotification confirming the design submission.

11. When the assay design results are ready, you receive an email notificationinstructing you to review the results in Ion AmpliSeq™ Designer. Click the Viewresults link provided in the email notification to be directed to the results page(or navigate to AmpliSeq.com, then click the notification or navigate to thecompleted design using the My Designs tab).

Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD RNA Gene Fusion design3



12. Review your order and order your panel.

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1 Switch design—Click the list to change the design in view.2 Edit—Edit the design name and details.3 Copy Targets—Copy your design to modify it.4 Start a new design—Start a new design.5 Chip Calculator—Use to obtain guidelines for sample library loading with various Ion chips to help in panel design.6 Add to cart—When this button is green you can add the highlighted design to your cart.





Chapter 3 Ion AmpliSeq™ HD custom panelsStart a new Ion AmpliSeq™ HD RNA Gene Fusion design 3


Oncomine™ tumor specific panels

■ Oncomine™ tumor specific panel designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

■ Workflow specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

■ Panel design screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

■ Price tiers for the Oncomine™ tumor specific panels . . . . . . . . . . . . . . . . . . . . . . 62

■ Order a core Oncomine™ tumor specific panel . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

■ Order a custom Oncomine™ tumor specific panel . . . . . . . . . . . . . . . . . . . . . . . . . 65

■ Oncomine™ tumor specific panel files . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

The Ion Torrent™ Oncomine™ tumor specific panel designs are small curated panelswith verified performance that complement the on-market Oncomine™ assays andprovide 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 inludes 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 performanceof each panel was verified on oncological research samples. 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.

4


Oncomine™ tumor specific panel designs

The following targeted Oncomine™ tumor specific panel designs are available in IonAmpliSeq™ Designer.

All Oncomine™ tumor specific panel designs have been laboratory tested on both low-quality (FFPE) and high-quality DNA controls.

IMPORTANT! The Oncomine™ tumor specific panels are for research use only.

Panel design Description

Oncomine™ Bladder Panel A 25-gene panel applicable to urothelial carcinoma research, whichrepresents ~90% of bladder cancers. Contains genes such asPIK3CA, FGFR3, and ERBB2. To view the targets that are included inthe panel, see “Oncomine™ Bladder Panel“ on page 80.

Oncomine™ BRCA Expanded Panel A 15-gene panel containing BRCA1 and BRCA2, as well as HRpathway genes important for ovarian, breast, and prostate cancerresearch. Contains genes such as ATM, PALB2, and BRIP1. To viewthe targets that are included in the panel, see “Oncomine™ BRCAExpanded Panel“ on page 81.

Oncomine™ Colorectal and Pancreatic Panel A 24-gene panel applicable to colorectal and pancreaticadenocarcinoma research. Includes DNA mismatch repair pathwaygenes and genes such as APC, KRAS, and NRAS. To view the targetsthat are included in the panel, see “Oncomine™ Colorectal andPancreatic Panel“ on page 82.

Oncomine™ Gastric and Esophageal CancerPanel

A 17-gene panel applicable to gastric, esophageal, andgastroesophageal adenocarcinoma research. Contains genes such asTP53, ERBB2, and CDKN2A. To view the targets that are included inthe panel, see “Oncomine™ Gastric and Esophageal Panel“ onpage 83.

Oncomine™ Gynecological Panel A 19-gene panel applicable to research of endometrial, cervical, andovarian carcinomas. Contains genes such as PTEN, BRCA2, andCTNNB1. To view the targets that are included in the panel, see “Oncomine™ Gynecological Panel“ on page 84.

Oncomine™ Kidney Panel A 15-gene panel applicable to research of renal cell carcinomas, themajority of kidney cancer. Contains genes such as VHL, MTOR, andPBRM1. To view the targets that are included in the panel, see “Oncomine™ Kidney Panel“ on page 84.

Oncomine™ Liver Panel A 22-gene panel applicable to hepatocellular carcinoma (HCC) andintrahepatic cholangiocarcinoma (ICC) research. Contains genes likeTP53, MYC, TERT, and CTNNB1. To view the targets that are includedin the panel, see “Oncomine™ Liver Panel“ on page 85.

Oncomine™ Lymphoma Panel A 25-gene panel applicable to research of non-Hodgkin’s lymphomas(primarily diffuse large B cell (DLBCL)) as well as to Hodgkin’slymphoma. Contains genes such as BCL2, MYD88, and CARD11. Toview the targets that are included in the panel, see “Oncomine™

Lymphoma Panel“ on page 86.

Chapter 4 Oncomine™ tumor specific panelsOncomine™ tumor specific panel designs 4


Panel design Description

Oncomine™ Melanoma Panel A 30-gene panel applicable to cutaneous and uveal melanomaresearch. Contains genes such as BRAF, NRAS, and CDKN2A. To viewthe targets that are included in the panel, see “Oncomine™ MelanomaPanel“ on page 87.

Oncomine™ Prostate Panel A 21-gene panel applicable to prostate adenocarcinoma research.Contains genes such as AR, PTEN, and MYC. To view the targets thatare included in the panel, see “Oncomine™ Prostate Panel“ onpage 88.

Workflow specifications

Oncomine™ tumor specific panels are compatible with the following workflow. Forend-to-end workflow details, see the Oncomine™ tumor specific panels User Guide(Pub. No. MAN0018518).

Workflow component Specification

Chip Ion 530™ Chip

Instrument Ion S5™ System, Ion S5™ XL System, IonGeneStudio™ S5 Series System[1]

Library preparation Ion AmpliSeq™ Library Kit PlusIon AmpliSeq™ Kit for Chef DL8

Samples per chip 8 (DNA)

Ion Reporter™ Software workflow Supported

BED files Provided

Copy number variant (CNV) baseline Supported

Oncomine™ Reporter Software Supported

[1] Unless otherwise specified, the Ion GeneStudio™ S5 Series System refers generically to the Ion GeneStudio™ S5 System, Ion GeneStudio™ S5 Plus System, and Ion GeneStudio™ S5 Prime System.

Chapter 4 Oncomine™ tumor specific panelsWorkflow specifications4


Panel design screen

You can review details about each Oncomine™ tumor specific panel and genes that areincluded in a panel in the panel design screen. You can also use the panel designscreen to customize a panel. For information on how to customize an Oncomine™

tumor specific panel, see “Order a custom Oncomine™ tumor specific panel“ onpage 65.

1 2 3 4 5

67

8

Figure 1 Panel design screen

1 Panel details.2 Click Customize to unlock a panel and customize the panel design (see “Order a custom

Oncomine™ tumor specific panel“ on page 65).3 Click Download Files to download a ZIP file that contains information such as gene lists,

amplicon pools, targeted hotspots, and gene coverage performance data (see “Oncomine™

tumor specific panel files“ on page 70).4 Expand the More Actions dropdown list, then click Export Targets to export a CSV file that

lists panel gene targets, or click Amplicon Distribution to view or download the ampliconsize distribution histogram for a design.

5 Click Add to Cart & Get Pricing to proceed with the purchase of your panel. You will beprompted to make workflow-specific selections, then re-directed to thermofisher.com toreview your cart and complete your purchase. For more information, see “Order a coreOncomine™ tumor specific panel“ on page 63.

6 The Price Tier varies depending on the panel that you select and can change when youcustomize the panel design to add or remove genes (see “Price tiers for the Oncomine™

tumor specific panels“ on page 62).7 Click on the gene name to view the gene details and visualize amplicons in the Integrative

Genomics Viewer (IGV). For more information, see Figure 2.8 In a locked state ( ) state, the panel design is finalized and ready to order. In an unlocked

state ( ), the panel design, name, and details can be customized. For more information,see “Order a custom Oncomine™ tumor specific panel“ on page 65.

Chapter 4 Oncomine™ tumor specific panelsPanel design screen 4



2 3 4

6

5

1

Figure 2 Gene details

1 Gene details.2 Panel details.3 Click Previous or Next to view the details for the previous or next gene in the selected

panel.4 Click Close to close the gene details screen and return to the panel design screen.5 Customize the IGV view by zooming in, zooming out, or showing or hiding labels.6 Click (Cutomize) in the row of a track within the IGV view to customize the name,

height, or color of the track or expand, collapse, or squish the track.

Price tiers for the Oncomine™ tumor specific panels

Oncomine™ tumor specific panels are priced according to whether the pre-curatedpanels are customized or not, as well as the number of genes in the panel design. The6 price tiers based on panel size are described in the following table. The price isconsistent within each tier. For example, a panel with 2 genes or 10 genes will havethe same Tier 1 pricing. For more information, see Table 2.

Price tier Number of genes

1 1–10

2 11–30

3 31–50

4 51–75

Chapter 4 Oncomine™ tumor specific panelsPrice tiers for the Oncomine™ tumor specific panels4


Price tier Number of genes

5 76–100

6 101–150

Contact your sales representative or account manager for price information.

Order a core Oncomine™ tumor specific panel

Select and order one of the wet-lab tested Oncomine™ tumor specific panel designsthat are available in Ion AmpliSeq™ Designer to use as-is.

• For a description of the available Oncomine™ tumor specific panels, see “Oncomine™ tumor specific panel designs“ on page 59.

• To view gene content of each design, see “Oncomine™ tumor specific panels: acomplete list of targets“ on page 80.

• To create and order a customized Oncomine™ tumor specific panel, see “Order acustom Oncomine™ tumor specific panel“ on page 65.

1. In the Home screen, in the Oncomine™ tumor specific panels pane, click Browsetumor types.

2. In the Oncomine™ tumor specific panels screen, click to select a panel of interest,then click Next.The panel design screen opens, where you can view panel details, downloadpanel-specific files, and customize the panel. For more information, see “Paneldesign screen“ on page 61.

3. Click Download Files, then in the Download Files dialog, click Download.

Note: You can only download files for finalized ( locked) designs.

A ZIP file that contains gene lists, amplicon pool information, amplicon sizehistogram, targeted hotspots, and gene coverage data is downloaded to yourlocal storage. For more information on file contents, see “Oncomine™ tumorspecific panel files“ on page 70.

Chapter 4 Oncomine™ tumor specific panelsOrder a core Oncomine™ tumor specific panel 4


4. (Optional) Click More Actions, then select one of the following options from thedropdown list.

Option Description

ExportTargets

Export the CSV file (<panel ID>_Oncomine_selected_targets.csv) that contains the list oftargets included in your design to your local storage.

AmpliconDistribution

View the amplicon size histogram for your design.To export the PNG file (<panel ID>_Oncomine_amplicon_size_histogram.png) that containsthe amplicon size histogram for your design to your local storage,click Download in the Amplicon Distribution window.

Note: The <panel ID>_Oncomine_amplicon_size_histogram.png is alsodownloaded as part of the ZIP file described in step 3.

5. Review your design, then click Add to Cart & Get Pricing.The Order Configuration window opens.

6. Make the appropriate selections for Panel, Workflow, and the number ofReactions, then click Add to Cart & Get Pricing.The respective Ion AmpliSeq™ library kit is added to your order.

7. In the Additional Products screen, review the items in your order and make anyadditional selections as needed by navigating through the Library Preparation,Templating and Sequencing, and Data Analysis workflow steps.

a. In the Library Preparation step, review the details for the panel design andthe library kit, then click Next.

b. In the Templating and Sequencing step, confirm your selection for thetemplate preparation platform, then if needed, select your sequencingplatform. Click Next.When you select a sequencing platform, a set of corresponding Ion Chip™

kits are added to your order. In the Qty column, in the row of the desiredIon Chip™ kit, enter the number of kits needed. Leave the Qty value at 0 forkits that you do not need.

Note: You can also remove or add kits when you review your cart on thermofisher.com.

8. Click Add to Cart on ThermoFisher.com, review the order specifications, thenclick Agree.You are redirected to the Cart on thermofisher.com, where you can add orremove items from your order as needed, view product pricing, and place yourorder.

The core Oncomine™ tumor specific panels are ready to use with the provided TorrentSuite™ Software planned run templates and Ion Reporter™ Software analysisworkflows.

Chapter 4 Oncomine™ tumor specific panelsOrder a core Oncomine™ tumor specific panel4




Order a custom Oncomine™ tumor specific panel

You can customize core Oncomine™ tumor specific panels by adding or removinggenes. You can also customize previously created and locked custom panels. The coreOncomine™ tumor specific panels are locked and ready to order. To customize alocked panel, you must first unlock the design. In an unlocked state, you can add orremove genes from the panel and edit the design name and details. When you finalizeyour customized panel design, you must lock the design in order to download panel-specific files, view amplicon distribution, and proceed with the purchase of yourpanel.

If you want to customize a previously created and locked custom design, click MyDesigns4Oncomine Tumor Specific, then in the Design column, click the name ofthe design of interest. In the panel design screen, click More Actions4Copy Design,then proceed to substep 3a.

1. In the Home screen, in the Oncomine™ tumor specific panels pane, click Browsetumor types.

2. In the Oncomine™ tumor specific panels screen, click to select a panel of interest,then click Next.The panel design screen opens, where you can view panel details , downloadpanel-specific files, and customize the panel design.

3. In the panel design screen, click Customize, then in the Customize window,enter panel name and details.

a. (Required) In the Name field, enter a unique name for your custom panel.The panel name can be up to 20 characters in length and must be unique.

b. (Optional) In the Details field, enter additional information for your custompanel.

4. Click Save.The selected panel design is unlocked and enabled for customization. Unlockeddesigns display next to the panel name in the panel design screen.

Chapter 4 Oncomine™ tumor specific panelsOrder a custom Oncomine™ tumor specific panel 4


5. Add or remove genes of interest.

Option Description

Add coregenes orassociatedresearchgenes

In the Core Genes section or in the Associated Research Genessection, use one of the following approaches to add one or moregenes to your new customized panel.

• Select a checkbox for each gene that you want to include in yourpanel (see callout 3 in Figure 3).

• Click Include All to select all of the displayed genes to beincluded in your panel.

Genes that are included in a panel are marked in green (see callout 1in Figure 3).

Add in-stockgenes

1. Click Add In Stock Genes (see callout 6 in Figure 3).The Add In Stock Genes window opens.

2. Manually add individual genes to your panel or input a list ofgenes of interest by file.

• In the Add Genes tab, enter a gene symbol for each genethat you want to add (for example, enter AKT1). If addingmore than one gene, separate each gene symbol by acomma, a space, or a new line (by pressing Return aftereach gene symbol).

• In Upload File tab, click Choose File, navigate to a CSV filethat contains a list of gene symbols for genes that you wantto add to your custom panel, then click Open.To download a template CSV file that you can copy/edit witha list of gene symbols of interest, click Download thisexample.

In-stock genes are added to the Added Genes section (see callout 5 in Figure 3).

Removegenes

In the Core Genes, Associated Research Genes, or Added Genessection, use one of the following approaches to remove one or moregenes from your custom panel.

• Deselect a checkbox for each gene that you want to exclude fromyour custom panel (see callout 3 in Figure 3).

• Click to remove a gene from a panel (see callout 4 in Figure 3).This action also removes a gene from the panel design screen. Ifyou remove a gene by mistake, you can add the gene back toyour custom panel using the Add In Stock Genes functionality.

• Click Exlude All to exclude all core genes or associated researchgenes from your custom panel.

Genes that are excluded from a panel appear greyed out (see callout2 in Figure 3).

Chapter 4 Oncomine™ tumor specific panelsOrder a custom Oncomine™ tumor specific panel4


1

2

6

3 4

7

5

Figure 3 Custom panel design screen

1 A gene that is included in the panel.2 A gene that is excluded from the panel.3 Select a checkbox to include a gene in the panel or deselect a checkbox to exlude a

gene from the panel.4 Click to remove a gene from a panel and the panel design screen.5 Added Genes section displays in-stock genes that have been added to the panel.6 Click Add In Stock Genes to add one or more in-stock genes to the panel.7 Click Finalize Design to finalize and lock the design.

6. Click Finalize Design to lock the design, then click Finalize Design in theconfirmation dialog (see callout 7 in Figure 3).It may take up to 30 minutes to finalize the design. When complete, the FinalizeDesign button is replaced by Add to Cart & Get Pricing.

7. Click Download Files, then in the Download Files dialog, click Download.

Note: You can only download files for finalized ( locked) designs.

A ZIP file that contains gene lists, amplicon pool information, amplicon sizehistogram, targeted hotspots, and gene coverage data is downloaded to yourlocal storage. For more information on file contents, see “Oncomine™ tumorspecific panel files“ on page 70.



8. (Optional) Click More Actions, then select one of the following options from thedropdown list.

Option Description

Edit DesignName &Details

(Unlocked custom designs only) Open the Edit Custom Designwindow and edit design name and details.

ExportTargets

Export the CSV file (<panel ID>_Oncomine_selected_targets.csv) that contains the list oftargets included in your design to your local storage.

AmpliconDistribution

View the amplicon size histogram for your design.To export the PNG file (<panel ID>_Oncomine_amplicon_size_histogram.png) that containsthe amplicon size histogram for your design to your local storage,click Download in the Amplicon Distribution window.

Note: The <panel ID>_Oncomine_amplicon_size_histogram.png is alsodownloaded as part of the ZIP file described in step 3.

Copy Design (Custom designs only) Copy a design.The design is added to the My Designs list in the Oncomine™ TumorSpecific tab.

Sharing (Locked custom designs only) Share a design.

Restore (Unlocked custom designs only) Restore a custom panel back to thecore design.

Delete (Custom designs only) Permanently delete a design.

9. After you review and finalize your design, click Add to Cart & Get Pricing.The Order Configuration window opens.

10. Make the appropriate selections for Panel, Workflow, and the number ofReactions, then click Add to Cart & Get Pricing.The respective Ion AmpliSeq™ library kit is added to your order.

11. In the Additional Products screen, review the items in your order and make anyadditional selections as needed by navigating through the Library Preparation,Templating and Sequencing, and Data Analysis workflow steps.

a. In the Library Preparation step, review the details for the panel design andthe library kit, then click Next.

Chapter 4 Oncomine™ tumor specific panelsOrder a custom Oncomine™ tumor specific panel4


b. In the Templating and Sequencing step, confirm your selection for thetemplate preparation platform, then if needed, select your sequencingplatform. Click Next.When you select a sequencing platform, a set of corresponding Ion Chip™

kits are added to your order. In the Qty column, in the row of the desiredIon Chip™ kit, enter the number of kits needed. Leave the Qty value at 0 forkits that you do not need.

Note: You can also remove or add kits when you review your cart on thermofisher.com.

12. Click Add to Cart on ThermoFisher.com, review the order specifications, thenclick Agree.You are redirected to the Cart on thermofisher.com, where you can add orremove items from your order as needed, view product price estimates, andplace your order.

For custom designs, you must download the <panel ID>_Oncomine_Designed.bed file for use in creating a custom Torrent Suite™ Softwareplanned run template and Ion Reporter™ Software analysis workflow that arerequired for templating, sequencing, and data analysis of each customized tumorspecific panel.





Oncomine™ tumor specific panel files

The following files are available for download for finalized ( locked) Oncomine™

tumor specific panel designs.

File Description

<panel ID>_Oncomine_amplicon_ insert_size_histogram.png

A PNG image of a histogram that represents the distribution of ampliconinsert sizes, excluding undigested forward and reverse primer sequencesand barcode adapters.

<panel ID>_Oncomine_amplicon_ size_histogram.png

A PNG image of a histogram that represents the distribution of ampliconsizes, including undigested forward and reverse primer sequences andbarcode adapters.

<panel ID>_Oncomine_coverage_ details.csv

A CSV file that contains exon-level coverage details, including target gene,number of amplicons that are required to cover the exon, and number ofbases covered by the amplicons.

<panel ID>_Oncomine_coverage_ summary.csv

A CSV file that contains gene-level coverage summary, including the totalnumber of amplicons required to cover the CDS region of the gene and totalnumber of bases covered by the amplicons.

<panel ID>_Oncomine_Designed.bed

A BED file that defines genomic coordinates that are targeted by the primers.

<panel ID>_Oncomine_hotspot.bed

A BED file that contains hotspot genomic coordinates that are covered by the <panel ID>_Oncomine_ Designed.bed file.

<panel ID>_Oncomine_Missed.bed

A BED file that contains coordinates that are missed by the designer; thedifference between submitted and designed.

<panel ID>_Oncomine_Submitted.bed

A BED file that contains genomic coordinates the primers are designed totarget.

plan.json A JSON file that contains information to automatically configure a run planfor a panel.

Chapter 4 Oncomine™ tumor specific panelsOncomine™ tumor specific panel files4


Supplemental information

■ Create a BED file from a list of variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

■ FASTA reference sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

■ Known polymorphism BED file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

■ Pipeline details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

■ Hotspots pipeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

■ Ion Chip capacities for Ion AmpliSeq™ DNA libraries sequenced atequal depths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

■ Ion Chip capacities for Ion AmpliSeq™ RNA libraries . . . . . . . . . . . . . . . . . . . . . 80

■ Oncomine™ tumor specific panels: a complete list of targets . . . . . . . . . . . . . . . 80

■ Frequently asked questions (FAQs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

■ Related Ion sequencing products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

■ Helpful tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

■ Advanced features and tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

Create a BED file from a list of variants

You can generate a custom BED file from the list of variants with your target regionsof interest using the UCSC Genome Browser, then import the BED file into IonAmpliSeq™ Designer.

To create a BED-formatted file starting from a list of dbSNP identifiers, use the UCSC Table Browser. Make sure to select genome and assembly as Human,(GRCh37/hg19).

Use the following example of creating a BED file from dbSNP targets with the UCSC Genome Browser to generate BED files from other variant formats, such asClinVar.

1. In the group list, select the Variation group .The track and table sections are populated with available SNP databases fromdbSNP.

A

Create a customBED file for inputinto IonAmpliSeq™

Designer

Start from a list ofdbSNP targetidentifiers


https://genome.ucsc.edu/

https://genome.ucsc.edu/cgi-bin/hgTables


2. In region, select the genome option.

3. In identifiers, click either paste list or upload list.

4. Either upload a text file listing your dbSNP identifiers, or simply paste in the list,then click submit. For the output format, select BED - browser extensible data.

5. Click get output,The Output as BED screen allows for padding with Whole Gene, Upstream orDownstream options.

Appendix A Supplemental informationCreate a BED file from a list of variantsA


The BED file appears similar to the following format.

This BED-formatted file can be uploaded to Ion AmpliSeq™ Designer.

1. Using the UCSC Table Browser, select these options.

Option Description

group Genes and Gene Prediction Tracks

track RefSeq Genes

table UCSC RefSeq (refGene)

output format BED - browser extensible data

Use the UCSCGenome Browserto create a BEDfile with paddedexons

Appendix A Supplemental informationCreate a BED file from a list of variants A



2. Upload or paste a list of gene identifiers. After you click submit, you are directedto the Output refGene as BED page.

3. Select options to add upstream or downstream base padding, or bases at eachend of the exons.

4. Select the appropriate options to create the BED file to include 5' or 3' UTRExons, or only coding exons.

FASTA reference sequence

Uploading sequences

One or more reference sequences in FASTA format can be uploaded using these steps.

1. Select a plain text or compressed file (in either ZIP or GZIP formats) that containsthe sequences. The maximum file size allowed for upload is 1 GB afterdecompression.

2. Click Enter FASTA data in a text area instead, then copy and paste the sequencesinto the available text area.

FASTA format

Appendix A Supplemental informationFASTA reference sequenceA


A reference sequence in FASTA format is expressed in 2 or more lines of text. The firstline is an identifying header, the remainder of the lines (one or more) represent thesequence itself.

• The header—The header line starts with a greater-than symbol (>) followed by atmost 64 ASCII characters. Allowed characters are A-Z, a-z, 0-9, "_" and "-", withNO SPACES between them. Because the header is used to identify the sequence,it is required to be unique for each sequence in the reference.

• The sequence—The only characters that are accepted for representing a sequenceare "A," "C," "G," "T," and "N". Lower case versions are also allowed forrepresenting low complexity regions. Although a sequence can be only one ormultiple lines of different sizes after the header, it is traditional to use separatelines of 50 or 60 characters in length.

• Sequence size—The minimum length of a sequence is 160 bp. Allowing 60 bp forminimum insert size, plus 50 bp upstream and 50 bp downstream to serve as adesign buffer for primer positioning during amplicon design;. However, therecommended upstream and downstream context buffer sequence for optimaldesigns is 1,000 bp.

Known polymorphism BED file

The known polymorphism BED file indicates regions of the sequences in the customreference FASTA file with high polymorphism (for example, SNPs, INDELs, or othervariations). Ion AmpliSeq™ Designer minimizes primer overlap with these regions.This file is optional. You can upload it at the time of creating a new custom reference.

The BED format is a tab-delimited file, with one line per region. Required fields arechrom, chromStart, and chromEnd in the first three columns of the BED file format.Additional fields are ignored.

The chrom field must match one contig ID in the accompanying FASTA file.chromStart and chromEnd fields are the zero-based, half-open coordinates indicatingthe region to target in the sequence identified by the ID in the chrom field. chromStartand chromEnd are relative to the sequence of the FASTA record corresponding to thegiven ID.

The chrom, chromStart, and chromEnd fields must meet the following criteria:• chromStart can be a value between 0 and length of the sequence specified by

chrom−1.• chromEnd must be greater than chromStart.• chromEnd can have a maximum value of the length of the sequence specified by

chrom.• No region should overlap any other region in the file. Combine overlapping

regions into a single contiguous region.• The variant coordinates BED file must have no header (for example, no "track"

lines).

Example FASTA file (50 bases per line):

Sample variants of interest (highlighted in blue).

Appendix A Supplemental informationKnown polymorphism BED file A


The following is a sample formatted BED file.

chrom chromStart chromEnd

contig_1 0 1

contig_1 95 96

contig_1 105 106

contig_1 199 200

Pipeline details

When you use Ion AmpliSeq™ Designer, the tiling and pooling algorithms run behindthe scenes to transform your submitted design into an orderable Custom IonAmpliSeq™ kit.

The following sections describe the Ion AmpliSeq™ tiling and pooling processes.

The pipeline is optimized to find the highest coverage with the least number ofamplicon pools. In an attempt to design all primer pairs exhaustively, the pipelinecreates an exhaustive search by sliding a tiling window one-base at a time across theentire targeted region.

Start End

The designpipeline

Appendix A Supplemental informationPipeline detailsA


In creating the primers, the pipeline considers:• A basic filtering

– Optimal melting temperatures– The manufacturability of the resulting primers (the resulting primers should

not contain long homopolymers)– The nucleotide composition should be between 20%-80% GC

• A biological filtering– SNP filtering: no known SNP (dbSNP 138) is allowed in a primer if its Minor

Allele Frequency (MAF) is greater than 5%. If MAF < 5%, SNPs are allowedanywhere in the primer.

• An off-target analysis to minimize off-target hybridization• An overlap analysis to reduce the number of primers (minimal tiling path),

because a higher overlap implies a higher cost

The tiling algorithm selects a subset (a tiling) of the input amplicons that meets thefollowing criteria.

• Covers as much of each target sequence as does the original input.• Has many fewer amplicons than the input set.• Maximizes the quality of the amplicons.

To do this, the tiling algorithm performs the following steps.

1. Creates an overlap graph of candidate inserts.2. Assigns a cost to each edge.3. Returns the tiles from the least-cost path from source to sink.

The output from the tiling algorithm is used as input for the pooling algorithm.

Improvements in the tiling algorithm have significantly increased the in silicocoverage of the designs. The users are enabled to choose between in silico and assayperformance.

The pooling algorithm assigns each tile (amplicon) to a pool, subject to requirementsthat allow each pool to be multiplexed.

To assign each tile to a pool, the pooling algorithm performs the following steps.

1. Sorts amplicons by decreasing priority.2. Starts with one available pool.3. Empties all available pools.4. For each sorted amplicon, the algorithm performs the following tasks.

a. If there is at least one pool in which the amplicon can be multiplexed, thealgorithm puts the amplicon into the first compatible pool that maximizes thedistance between the amplicon and any other amplicon in the pool.b. If a new pool can be added, the algorithm adds a new available pool andreturns to step 3.c. If a new pool cannot be added, the algorithm puts the amplicon into theoverflow (unpooled) list.

Tiling algorithm

Pooling algorithm

Appendix A Supplemental informationPipeline details A


• The tiling algorithm ensures that the PCR pools cover as much of the targets asdo the candidate amplicons supplied to it.

• The pooling algorithm generates a very small number of PCR pools.• The primers in a pool do not interact.• Everything runs quickly and with reasonable memory resulting in faster design

creation.

Hotspots pipeline

Single pool panel designs represent cost and handling advantages over panels thatrequire two or more pools of primers. Designs targeting small genomic regions suchas SNPs and small INDELs are well suited to fit in a single pool because those regionscan be covered by non-overlapping amplicons.

In standard designs, the "tiling" stage of the pipeline is when amplicons are selected tocover the target regions. Because the regions are often larger than a single amplicon,several amplicons are required to cover one particular target. This process results in aset of overlapping amplicons that, if put together, are prone to interact with each otherduring the sequencing process. The "pooling" stage is when as many pools as neededare created to avoid these interactions.

The hotspots pipeline includes special algorithms that allow in the pooling stage thecreation of a single pool of primers with maximum coverage for targets as small asone base, and as large as 50 bases.

The idea behind the hotspots designer can be better understood by looking at thefollowing diagram in which the long line represents a genomic region; a, b, and c arethe amplicons available for covering that region. The red portions of the ampliconsrepresent the primers.

cb

a

In a standard (non-hotspots) Ion AmpliSeq™ panel, the targets are long regions(delimited by arrows in the diagram). The algorithm attempts to find a set of primersat minimum cost to cover the target (amplicons a and b). Amplicons a and b overlap,so this would require two pools to cover the region in question. Forcing the panel tobe in one pool would eliminate amplicon a or b resulting in reduced coverage for theone-pool solution. Using amplicons c and b is not an option because they fail to covera portion of the region between the arrows. In contrast, for a hotspots panel, thetargets are small genomic regions (marked with dots over the line representing thegenome), the hotspots pipeline would actually select non-overlapping amplicons cand b to cover the submitted targets. Non-overlapping amplicons can be put togetherin a single pool of primers.

Advantages ofpooling and tiling

Appendix A Supplemental informationHotspots pipelineA


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

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

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

Chip Ion 314™ Ion 316™

Ion 510™Ion 318™

Ion 520™ Ion 530™ Ion PI™

Ion 540™ Ion 550™

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

Expectedcoverage

95%>30X

95%>500X

95%>30X

95%>500X

95%>30X

95%>500X

95%>30X

95%>500X

95%>30X

95%>500X

95%>30X

95%>500X

Number ofamplicons per

libraryApproximate number of libraries per chip

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

Appendix A Supplemental informationIon Chip capacities for Ion AmpliSeq™ DNA libraries sequenced at equal depths A


Ion Chip capacities for Ion AmpliSeq™ RNA libraries

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

For panels containing fusion detection primer pairs, higher library multiplexing ispossible because most targets are not present, and therefore do not create librarymolecules. For most fusion detection assays, only ~250,000 reads per library arerequired.

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

Number of libraries sequenced per chip = Chip capacity in reads

(Sequencing depth) (Number of primer pairs)

These recommendations serve as suggestions and the actual capacity to multiplexlibraries is determined by the expression levels of the genes included in your IonAmpliSeq™ RNA panel. The expression levels of the individual genes can vary byinput RNA type. We suggest using the formula for new panels and determiningactual multiplexing limits empirically.

Ion Chip

Ion 314™

ChipIon 316™

ChipIon 510™

Chip

Ion 318™/Ion 520™

Chip

Ion 530™

Chip

Ion PI™/Ion 540™

Chip

Ion 550™

Chip

Chip capacityin reads (M)

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

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

Oncomine™ tumor specific panels: a complete list of targets

The following targets are included in the Oncomine™ Bladder Panel design.

Target name Design type

AKT1 Hotspot

ARID1A Full CSD

ATM Gene

BRAF Hotspot

Oncomine™

Bladder Panel

Appendix A Supplemental informationIon Chip capacities for Ion AmpliSeq™ RNA librariesA



CCND1 Hotspot

CCNE1 Hotspot

CDKN1A Gene

CDKN2A Gene

CTNNB1 Hotspot

E2F3 Gene

ERBB2 Hotspot

ERBB3 Hotspot

ERCC2 Gene

FGFR2 Hotspot

FGFR3 Hotspot

HRAS Hotspot

KDM6A Gene

KRAS Hotspot

MDM2 Gene

PIK3CA Gene

PPARG Gene

PTEN Gene

RB1 Gene

TP53 Gene

TSC1 Gene

The Oncomine™ BRCA Expanded panel supports research into the homologousrecombination repair pathway. The following targets are included in the Oncomine™

BRCA Expanded Panel design.


ATM Gene

BARD1 Gene

BRCA1 Gene

BRCA2 Gene

BRIP1 Gene

Oncomine™ BRCAExpanded Panel

Appendix A Supplemental informationOncomine™ tumor specific panels: a complete list of targets A



CDK12 Gene

CHEK2 Gene

FANCD2 Gene

MRE11 Gene

NBN Gene

PALB2 Gene

PPP2R2A Gene

RAD51B Gene

RAD54L Gene

TP53 Gene

The following targets are included in the Oncomine™ Colorectal and Pancreatic Paneldesign.


APC Gene

ARID1A Gene

BRAF Hotspot

CDKN2A Gene

CTNNB1 Hotspot

ERBB2 Hotspot

ERBB3 Hotspot

FBXW7 Gene

GNAS Hotspot

KRAS Hotspot

MLH1 Gene

MSH2 Gene

MSH6 Gene

MYC Gene

NRAS Hotspot

PIK3CA Gene

PMS2 Gene

Oncomine™

Colorectal andPancreatic Panel

Appendix A Supplemental informationOncomine™ tumor specific panels: a complete list of targetsA



POLE Gene

PTEN Gene

RNF43 Gene

SMAD4 Gene

SOX9 Gene

TCF7L2 Gene

TP53 Gene

The following targets are included in the Oncomine™ Gastric and Esophageal Paneldesign.


APC Gene

ARID1A Gene

CDKN2A Gene

CTNNB1 Hotspot

ERBB2 Hotspot

ERBB3 Hotspot

FBXW7 Gene

GNAS Hotspot

KMT2C Gene

KMT2D Gene

KRAS Hotspot

MYC Gene

PIK3CA Gene

PTEN Gene

RNF43 Gene

SMAD4 Gene

TP53 Gene

Oncomine™

Gastric andEsophageal Panel



The following targets are included in the Oncomine™ Gynecological Panel design.


AKT1 Hotspot

ARID1A Gene

BRCA1 Gene

BRCA2 Gene

CCNE1 Hotspot

CTNNB1 Hotspot

ERBB2 Hotspot

ERBB3 Hotspot

FBXW7 Gene

FGFR2 Hotspot

KRAS Hotspot

MYC Gene

PIK3CA Gene

PIK3R1 Gene

POLE Gene

PPP2R1A Hotspot

PTEN Gene

RB1 Gene

TP53 Hotspot

The following targets are included in the Oncomine™ Kidney Panel design.


ATM Gene

BAP1 Gene

KDM5C Gene

MET Hotspot

MTOR Hotspot

NF2 Gene

PBRM1 Gene

PIK3CA Gene

Oncomine™

GynecologicalPanel

Oncomine™ KidneyPanel




PTEN Gene

SETD2 Gene

SMARCB1 Gene

TP53 Gene

TSC1 Gene

TSC2 Gene

VHL Gene

The following targets are included in the Oncomine™ Liver Panel design.


ALB Gene

APOB Gene

ARID1A Gene

ARID2 Gene

AXIN1 Gene

CCND1 Hotspot

CDKN2A Gene

CTNNB1 Hotspot

IDH1 Hotspot

IDH2 Hotspot

JAK1 Gene

KEAP1 Gene

KRAS Hotspot

MYC Gene

NFE2L2 Hotspot

PIK3CA Gene

RB1 Gene

RIT1 Gene

RPS6KA3 Gene

TERT Gene

Oncomine™ LiverPanel




TP53 Gene

TSC2 Gene

The following targets are included in the Oncomine™ Lymphoma Panel design.


ARID1A Gene

ATM Gene

B2M Gene

BCL2 Gene

BCL6 Gene

BRAF Hotspot

BTK Hotspot

CARD11 Hotspot

CD79B Hotspot

CDKN2A Gene

CREBBP Gene

EZH2 Gene

GNA13 Gene

HIST1H1E Gene

KMT2D Gene

MTOR Hotspot

MYC Gene

MYD88 Hotspot

PIM1 Gene

SF3B1 Hotspot

SOCS1 Gene

TNFAIP3 Gene

TNFRSF14 Gene

TP53 Gene

XPO1 Hotspot

Oncomine™

Lymphoma Panel



The following targets are included in the Oncomine™ Melanoma Panel design.


AKT3 Hotspot

ARID2 Gene

BRAF Hotspot

CCND1 Hotspot

CDK4 Gene

CDKN2A Gene

CTNNB1 Hotspot

ERBB4 Hotspot

EZH2 Gene

GNA11 Hotspot

GNAQ Hotspot

GRIN2A Gene

HRAS Hotspot

IDH1 Hotspot

KIT Hotspot

KRAS Hotspot

MAP2K1 Hotspot

MDM2 Gene

MITF Hotspot

NF1 Gene

NRAS Hotspot

PIK3CA Gene

PPP6C Gene

PTEN Gene

RAC1 Hotspot

RB1 Gene

TERT Gene

TP53 Gene

TYR Gene

Oncomine™

Melanoma Panel



The following targets are included in the Oncomine™ Prostate Panel design.


AKT1 Hotspot

APC Gene

AR Hotspot

BRAF Hotspot

BRCA2 Gene

CDK12 Gene

CTNNB1 Hotspot

FOXA1 Hotspot

HRAS Hotspot

IDH1 Hotspot

KDM6A Gene

KMT2D Gene

KRAS Hotspot

MED12 Hotspot

MYC Gene

PIK3CA Gene

PIK3R1 Gene

PTEN Gene

RB1 Gene

SPOP Hotspot

TP53 Gene

Oncomine™

Prostate Panel



Frequently asked questions (FAQs)

1. How does the software accommodate intronic regions?When you submit a gene to design, only exons are used as targets. To designacross the whole gene (exons and introns), submit the start and end coordinatesof the gene.

2. When I enter gene symbols, does the design include promoter regions?No. The designer uses exon coordinates as listed by the UCSC Genome Browser.Promoters are not part of the exons and are requested using a BED file describingthe genome coordinates.

3. What is the level of overlap among the primers? Are the overlapping primersin the same tube?Primers in the same tube do not overlap. As our product line evolves in thefuture, a small overlap might be possible.

4. For Ion AmpliSeq™ Designer, are primer sets designed automatically (with acomputer program), without interrogation from a research scientist?The process is an automated pipeline, optimized to provide the maximumcoverage with reliable primer sets.

5. How are the Ion AmpliSeq™ Custom designs verified?Each primer pool goes through a rigorous process to meet strict designspecifications. During the design of our pipeline, we verified a substantialnumber of our custom assays though wet lab testing.

6. Can I use a subset of the Fixed Panels for a custom design?Yes. Clicking the Customize Panel button for the Panel design you are workingwith creates a starting template where you can delete or add genes or regions.

7. For 200-bp designs, should the BED file that is submitted be in a 175−225-bprange?No, you do not have to select the gene coordinates (BED file) to be 175−225 bp.Ion AmpliSeq™ Designer designs the primer pairs, and provides the appropriateBED file and primer sequences to generate amplicons approximately 200-bp inlength.

8. If I submit two continuous regions (175−225-bp range each) combined as oneBED file, is it possible to get the designed primers for the overlapping region?If overlapping regions are submitted to the design pipeline, internally the regionis concatenated and treated as a single region for design, therefore there is nooverlap. The two regions are reported back in the UI as submitted. Although it ispossible that an amplicon is prorated twice, one time in each original region, thisamplicon (and its primers) only occurs one time in the design (see the plate file).

9. The PDF report from my Ion AmpliSeq™ Cancer Panel runs shows that for thealigned sequence section, we have 0.00% coverage of the genome. Is this resultbecause the fraction of the genome that is covered is so low compared to thehg19 reference that we are not showing sufficient decimal places?If a full reference was used, and you have only tens of Mbs, the coverage is solow that we do not track this low of a percentage.

10. What is a superamplicon?A superamplicon is created when two forward PCRs combined to form one largeamplicon. The pooler algorithm in the pipeline separates primers into separatepools to minimize this occurrence.

Ion AmpliSeq™

Designer generalFAQs

Appendix A Supplemental informationFrequently asked questions (FAQs) A


11. The BED file specifications state that in a BED file the chrStart number is zero-indexed and the chrEnd number is not included in the feature. Are youfollowing this convention for upload and are the numbers shown in thedesigner 1-indexed or 0-indexed?chromStart - The starting position of the feature in the chromosome or scaffold.The first base in a chromosome is numbered 0.chromEnd - The ending positionof the feature in the chromosome or scaffold. The chromEnd base is not includedin the display of the feature. For example, the first 100 bases of a chromosome aredefined as chromStart=0, chromEnd=100, and span the bases numbered 0-99.

12. Can you describe further how the Ultraplex technology works?Development work from over a decade allows us to produce primer designs thatallow simultaneous amplification of many amplicon targets. A unique chemistryhas been developed for Ion AmpliSeq™ libraries that allows removal of anyprimer-dimer that is formed, along with most of the primer itself from theamplified template. This removal makes sequencing efficient by not wastingbases on non-informative primer sequence, and allows for clean sequencingreactions.

13. Do your designs account for the presence of pseudogenes?Yes. The pipeline first attempts to design primers that only match the target, andnot the pseudogene (or duplicate) versions. If the target gene is not covered inthe first rounds of primer selection, then the match parameters are relaxed, forthe sake of coverage, in later rounds, attempting to maintain the uniqueness ofthe inserts.

14. If two amplicons overlap, do the primers produce a large product in additionto two small products?The pooling step in the design is optimized to minimize the interference betweenoverlapping amplicons. Overlapping amplicons are segregated into differentpools.

15. Why is my gene not accepted for design?There are several reasons why your gene is not accepted:

a. A gene must be part of the UCSC Reference Gene dataset.b. A gene must have at least one coding transcript.c. A gene must not map to more than one genomic location (including

pseudoautosomal genes (PAR1,2).d. A gene must not map to unassembled contigs or alternate assemblies -

examples for human include: chrUn_gl000228, chr4_gl000194_random andchr6_cox_hap2.

16. Which sequence versions does Ion AmpliSeq™ Designer use in itscomputations?DNAHuman Genome* - Feb. 2009 (hg19, GRCh37), Mouse Genome* - Dec. 2011(mm10, GRCm38), Gene targets correspond to RefSeq v63, Hotspots targetscorrespond to dbSNP v138 (for human and mouse) and COSMIC v68 (availableonly for human), RNAHuman RNA Canonical RefSeq Transcripts* - Feb. 2009(hg19, GRCh37), HGNC Database, HUGO Gene Nomenclature Committee(HGNC), EMBL Outstation - Hinxton, European Bioinformatics Institute,Wellcome Trust Genome Campus, Hinxton, and Cambridgeshire, CB10 1SD, UKhttp://www.genenames.org, 11/2012

Appendix A Supplemental informationFrequently asked questions (FAQs)A


General

1. What sample types are compatible with Oncomine™ tumor specific panels.Oncomine™ tumor specific panels are designed to be compatible with degradedDNA such as FFPE, but can also be used on FNA (fine needle aspirate), freshfrozen samples, and other high quality DNA.

2. What is the price? How is pricing determined?Pricing is based on the panel configuration: manual vs Ion Chef™ librarypreparation, reaction pack size, and number of genes in the panel.

Table 2 Oncomine™ tumor specific panel pricing information

Panel type Core Custom

Library preparationmethod

Manual Ion Chef™ Manual Ion Chef™

# of reactions 24 96 384 32 128 24 96 384 32 128

1–10 genes N/A

11–30 genes

31–50 genes N/A

51–75 genes

76–100 genes

100–150 genes

3. I don't see Oncomine™ tumor specific panels in the Chip Calculator. Howmany samples can I load on a single chip?The Ion 530™ Chip is supported. The following table provides chip capacityestimates based on the number of genes in a DNA panel.IMPORTANT! The chip capacity information that is provided in this table is based on35 amplicons per gene, maximum number of genes per pricing tier, and 2,000X depth.

Sequencing system Ion GeneStudio™ PGM™ Genexus™

Ion Chip™ 510 520 530 540 550 316 318 GX5™

Reads (M) 2.5 4 17.5 70 120 3 5 49

1–10 genes 2 3 16 66 114 2 4 46

11–30 genes 1 1 8 33 57 1 2 23

31–50 genes – 1 5 20 34 – 1 14

51–75 genes – – 3 13 22 – – 9

76–100 genes – – 2 10 17 – – 7

100–150 genes – – 1 6 11 – – 4

Oncomine™ tumorspecific panelsFAQs



Panels and panel content

1. Being Oncomine™-branded, can I do lot matching?You should expect each order of a panel to be a new lot number. However, thereis a possibility that 2 orders provided on the same SO could be the same lotnumber.

2. I see some tumor types are missing, will other tumor types be addedeventually, such as brain or sarcoma?We are investigating the addition of new tumor types and new genes in thefuture. Please contact Support or provide assay feedback on AmpliSeq.com forany additions you would like to see, including additional genes. To providespecific design feedback, click More Actions4Give Solution Feedback in thepanel design screen (see callout 4 in Figure 1). To provide general feedback orrequest help, click the Assay Design Support link in the footer of each page on AmpliSeq.com.

3. What is the difference between a core panel and a custom panel?All genes in the inventory have been tested to ensure they perform well togetherin any permutation, core or customized. The core panels (tumor specific panels)and genes have been tested to the highest quality requirement compared to othergenes in the inventory as these will be most relevant to clinical research. Inaddition, each core panel had rigorous testing done on FFPE samples andpositive controls to set realistic expectations for panel performance.

4. What is the maximum and minimum number of genes and ampliconsallowed?The minimum number of amplicons is 24 (12 per pool). The maximum number ofamplicons is 5,000 or 150 genes, whichever is reached first.

5. The added library kits have different product numbers than I’m used to. Arethese product numbers different than the ones available on thermofisher.com?The library kits are the same as those commercially available, simply under adifferent product number. These library kits are only available with theOncomine™ tumor specific panels from thermofisher.com.

6. What rationale can you provide for the core panel gene content? What areassociated research genes?Core panel content is designed to be most relevant for clinical research of thetumor type based on drug labels, guidelines, and clinical trials. The panel can becustomized if you prefer to add or replace certain genes.Associated research genes can also be of interest based on our curation ofrelevant literature.

7. Can you differentiate the B cell and T-cell origin with the Lymphoma panel?Not currently. When a future panel includes targets for both cell types, you willbe able to determine the cell type of origin.

8. Why are the genes that I expect to see on the lymphoma panel not there?We have omitted all of the fusion (RNA) genes from this panel design. Supportfor fusions will be added in a future release. Even if there is a relevant (DNA)mutation on one of the fusion genes, it is omitted, so there is no assumption thatthe RNA component is included in the panel design. For instance, ALK fusionsare relevant, as are ALK mutations. ALK is omitted from this version of the paneldesign.







Ion AmpliSeq™ Designer

1. What does the hotspot track indicate in the Integrative Genomics Viewer(IGV)?Hotspots are regions in the genome where the risk for biologically relevantgenetic mutations is elevated (eg. INDELs and SNPs). Internally, we have curateda list of relevant hotspots that are annotated in Ion Reporter™ Software.Amplicons are designed to optimally cover these hotspots. Some genes haveamplicons that cover the entire gene, while other genes have amplicons that aredesigned to cover all relevant hotspots, but not necessarily the entire gene. Thenew track on AmpliSeq.com displays hotspots and their expected ampliconcoverage.

2. Where is the Spike-in feature of AmpliSeq On-Demand? What do I do if mygene is not present?We do not currently support a spike-in function for the Oncomine™ tumorspecific panels. All genes have been designed with respect to each other in orderto enable high performance panels, core or customized. We cannot guaranteeperformance of the panel when a MTO spike-in is used. If your gene is notpresent, please contact Support and we will attempt to include it in the nextphase of genes. An assay feedback tool is available in the Ion AmpliSeq™

Designer – submit requests to add new genes to inventory.3. What if I just want amplicons to cover hotspots for a certain gene, not the full

gene design?Each gene is available as-is without modification. Please contact Support for helpwith designing amplicons that cover only your genomic location of interest forinclusion into a spike-in. An assay feedback tool is available in the IonAmpliSeq™ Designer – submit any requests for modification to a gene design

4. What does design finalization mean?Design finalization involves generation of all of the final panel files. Somedesigns can take longer than 30 minutes. Contact Support if the process is notcomplete within a couple of hours.

Panel design and performance

1. What is the quality guarantee?Each gene was tested on FFPE samples in the context of all inventoried genes,and in the context of tumor-specific panels where appropriate to ensureperformance in any panel environment. However, since full customization ispossible, not every possible panel permutation was tested. Therefore, some geneand amplicon variability should be expected. Each tumor-specific panel hadadditional testing. We strive to achieve a high product quality to provide youwith consistent and reliable panel performance, please contact us if you havequestions or concerns.

2. Why does this product have an Assessment and Development packages butother Oncomine™ panels do not? Will these be availabe in the future?As a custom product, each panel order is considered a new lot. We recommendtesting each new lot to make sure it meets expectations. To support this effort, weare providing an assessment package which includes normal genomic DNA and




FFPE DNA to assess panel coverage and sequencing depth uniformity, as well asa positive control to assess preliminary performance.We have heard of the need for the ability to customize an Oncomine™ panel andrun an Oncomine™ panel on a different chip and maintain confidence in panelperformance. We have responded with the Development Package. This packagecontains sufficient normal genomic DNA and FFPE DNA for SVB and CNBgeneration, respectively, as well as a positive control to assess CNV calling.

Analysis and workflow

1. Can I detect copy number changes? Which genes?All genes, whether CDS or hotspot, have been designed with sufficientamplicons to enable CNV detection. BTK, a hotspot-only gene, is the onlyexception.

2. What is the supported workflow?The Oncomine™ tumor-specific panels are supported for use with the Ion 530™

Chip on the Ion GeneStudio™ S5 Series instruments. Manual library preparationis supported with the Ion AmpliSeq™ Library Kit Plus and Ion Torrent™ DualBarcodes. Ion Chef™ library preparation is supported with the Ion AmpliSeq™ Kitfor Chef DL8 and IonCode™ Barcode Adapters. Dedicated analysis workflows forthe Oncomine™ tumor specific panels are available in Ion Reporter™ Software.Generation of reports is supported in Oncomine™ Reporter Software.

3. Can I run a tumor specific panel on a chip other than Ion 530™ Chip? What do Ineed to make sure I can call SNPs/INDELs or CNVs?Yes, you can run a tumor specific panel on a non-Ion 530™ Chip. You will need tocreate a custom CNV baseline, SVB (sequence variant baseline), and hotspot file,then upload to a custom Ion Reporter™ analysis workflow. For the detailedprotocol, see the Oncomine™ tumor specific panels User GuideMAN0018518. Ifneeded, contact your FAS or FBS representative for support.

4. Can I run a customized panel on an Ion 530™ Chip? What do I need to makesure I can call SNPs/INDELs or CNVs?Yes, you can run a customized tumor specific panel on an Ion 530™ Chip. Youwill need to create a custom Ion Reporter™ analysis workflow using the BED filefrom AmpliSeq.com as well as create a custom CNV baseline. For the detailedprotocol, see the Oncomine™ tumor specific panels User GuideMAN0018518. Ifneeded, contact your FAS or FBS representative for support.

5. When I run CNV analysis in Ion Reporter™ Software, there is a CNV detectedat chr3:193207277 that is not associated with a gene. Is this real?Oncomine™ tumor specific assay analysis results can indicate a CNV atchr3:193207277 of 116bp in length and not associated with a gene. It is not a CNVhotspot but a sample ID amplicon and should be disregarded.

Using the Oncomine™ BRCA workflow in Ion Reporter™ Software 5.10 or IonReporter™ Software 5.12 to detect exon deletions in BRCA1 and BRCA2

1. I am used to running Oncomine™ BRCA panel. Is the analysis for Oncomine™

BRCA Expanded panel different? How do I get exon-level CNV?You can detect exon-level CNV in the Oncomine™ BRCA Expanded panel byanalyzing your sample using the Oncomine™ BRCA Ion Reporter™ workflow inIon Reporter™ Software 5.10 or later. You should first run the Oncomine™ BRCAExpanded workflow to detect SNPs, INDELs, and gene level CNVs in all thegenes in your panel. After that, you should run the Oncomine™ BRCA workflow




to examine the exon deletions in BRCA1 and BRCA2. We are working tointegrate the two workflows into a single workflow in a future version of the IonReporter™ Software that will launch after Ion Reporter™ Software 5.12.

Note: CNV baselines can always be augmented with your own samples toincrease the number of samples that pass quality control.

2. Why are some of the variants detected in BRCA1/2 in the Oncomine™ BRCAworkflow different from those detected in the Oncomine™ BRCA Expandedworkflow?The Oncomine™ BRCA Expanded workflow is optimized to the tumor-specificpanel and includes the analysis of all the genes in the panel. In contrast, theOncomine™ BRCA workflow only analyzes BRCA1 and BRCA2.

3. Why does the on-target look low when I use the Oncomine™ BRCA workflow?The Oncomine™ BRCA workflow only analyses BRCA1 and BRCA2 . Therefore,the on-target reported is artificially low because it ignores all the amplicons thatcome from the other genes in the BRCA Expanded panel or your custom panel.Use the On-target number reported in the Oncomine™ Expanded workflow oryour custom workflow.

4. Why are the rest of the genes in the panel missing when I use the Oncomine™

BRCA workflow?The Oncomine™ Expanded workflow is optimized to the tumor-specific paneland includes the analysis of all the genes in the panel. In contrast, theOncomine™ BRCA workflow only analyzes BRCA1 and BRCA2 and only showsSNPs, INDELs, and CNVs in those two genes.



General Ion AmpliSeq™ On-Demand Panel design questions

1. Why can I order only 500 genes in an On-Demand panel?For this version of the software, we have set an ordering limit to 500 genes or15,000 amplicons per panel due to manufacturing restrictions. As we continue tomake improvements this limit is likely to increase and larger designs may beordered.

2. Why is there a limit on the number of genes that I can add to my On-Demandpanel?Because the order limit is set at 500 genes per panel, it becomes impractical toallow a large number of genes into the Grid or Table view, which need to bedeselected to make the design orderable. For this reason we have introduced alimit on the number of genes that can be added to an On-Demand panel.

3. Can I edit the content after I have created a design?Yes, an On-Demand design can be edited after it has been created as long as ithas not been ordered, or a Spike-in Panel created. This is different from Made-to-Order designs, which can only be edited in the "Draft" mode, and become lockedafter the job has been submitted and the Results reported.

4. Can I download my list of targets after I have created a design?Yes, select the "Export targets" button to download the list as a CSV file. Thisexports all the targets that are displayed in the user interface (Selected,Deselected, Added).

5. After I've created a design, can I add more content from a Disease ResearchArea (DRA)?Not directly. Currently we do not allow addition of DRA content to an existingdesign, only to new designs. The workaround is to create a new design with thedesired DRA content, and then export the list of targets. You can then upload thislist to the desired existing On-Demand design.

6. What are the genes that are ordered when I click the “Order” button?When you click the "Order" button, only genes that are available as On-Demandgenes, and which you select are ordered. If you create a Spike-in Panel, that panelneeds to be ordered separately by visiting the results page of that panel.

7. Can I edit my design after I have placed an order?No, after you have placed an order, the design cannot be edited because thenecessary files needed for analysis by Torrent Suite™ and Ion Reporter™ Softwareneed to remain in sync with the material you ordered. If you need to edit yourdesign, select the "Clone" option. A new IAD number is then assigned to yourdesign, and you have the option to edit the design content.

8. Can I reorder a design after I’ve placed an initial order?Yes, you can always go back to your ordered design and place a new order.

9. What is the annotation source and version that is used to recognize genesymbols when creating an On-Demand Panel?The source of annotations is refGene, and the version that we are using is versionv74.

10. Are untranslated regions (UTRs) included with an On-Demand gene?No, only the coding DNA sequence (CDS) region of a gene is included as part ofan On-Demand gene design.

11. Are UTR-only genes supported? What about pseudogenes?No, only genes containing CDS regions are supported. At this time, pseudogenesare not supported.

Ion AmpliSeq™

On‑DemandPanels FAQs



12. What is the padding used for On-Demand gene designs?The padding for every On-Demand gene design is 5 bp on the 5’ and 3’ ends.

13. Can I share my design with a collaborator the same way I do with a Made-to-Order design (also known as Custom designs)?We currently do not support an easy share mechanism for sharing the designwith collaborators. However, you can export the list of targets, and share that listwith your collaborator. The design they create will be identical to yours if the listof targets is the same.

14. What is “in silico” coverage?“In silico” coverage is defined by the percentage of bases that are covered by thetiling of amplicons. This number is a computer-based calculation and should notbe confused with experimental coverage, which represents the actualperformance of the panel in the lab.

15. What is "Gene uniformity"?The number of reads spanning is counted for each base across all padded codingexons of a gene. An average value is calculated for all the bases, and thepercentage of bases with read counts above 20% of the average value is definedas "Gene uniformity".

16. Have you checked for all possible gene combinations to test for primer-primerinteractions?No, the number of possible combinations is astronomical and it is not possible totest for all possible combinations in the lab. What the team has done is usecomputer-based searches to reduce as much as possible the occurrence of primer-primer interactions. In addition, the genes have been synthesized in large genebatches, and we have observed less than 1% amplicon drop-out due to suspectedprimer-primer interactions.

Disease Research Areas (DRAs)

1. What are the sources used for creating the associations for the various DiseaseResearch Areas in the tool?The sources include DisGeNET (http://www.disgenet.org), Unified MedicalLanguage System (https://www.nlm.nih.gov/research/umls/) and MedicalSubject Headings (MeSH).

2. What algorithm was used to create such associations?An in-house gene scoring algorithm was used to create these associations. Detailsof the algorithm are proprietary.

3. What does the "Score" mean?The "Score" ranks the relationship between for researching the correlationbetween a gene and a disease. It takes into account both the strength and numberof gene-disease pairs.

4. Can I preview the content of a DRA before creating a design?No, a preview of the gene content is not available at this time. You need to createthe design to view the gene content.

5. Can I pre-select the gene content of a DRA before creating a design?No, gene content cannot be pre-selected. You can only select full DRA categoriesby clicking the box on the right, and then edit the gene content once the design isin the On-Demand Grid or Table views.

6. What is the number in parentheses next to each DRA?The number in parentheses ( ) denotes number of genes in the group.



http://www.disgenet.org

https://www.nlm.nih.gov/research/umls/

7. The gene count doesn’t seem to add up. Why is that?Gene counts often do not add up as the sum of the subcomponents because oneor more genes can belong to multiple DRAs.

8. My favorite gene is not present in a particular DRA. Why is that?Genes are scored based on their degree of association to a particular DRA by ouralgorithms that have aggregated the data. If your gene is not present, it is likelybecause the observed associations are below our threshold, or outside of thesources we used. Contact our support team ([email protected]) if you are aware of strong evidence demonstratingthat a gene should be included in a specific category.

9. What are "ACMG Recommendations…"?American College of Medical Genetics and Genomics (ACMG)Recommendations for Reporting of Incidental Findings in Clinical Exome andGenome Sequencing.

10. What are "Newborn Screening Conditions" or "Newborn Screening" genes?These are genes associated with conditions listed in the Recommended UniformScreening Panel (RUSP).

Integrative Genomics Viewer (IGV)

1. What is the "Expected coverage" track in the IGV?The "Expected coverage" track reflects the number of reads that were observedfor each amplicon of each targeted gene during our verification experiments.This track should only be used as general guidance of the likely performanceobserved when running the experiment. Values are likely to be different when anew assay is performed, but the general coverage trend should remain.

2. What are "Missed regions (if any)"?The "Missed regions" are regions where tiling of a high specificity amplicon wasnot possible due to local environment complexity. We have made every effort tominimize the occurrence of these regions in our On-Demand designs.

3. What is the scale on the Y-axis?The Y-axis represents the experimental coverage, which has been normalized to100.

4. Can I use coordinates to navigate the IGV viewer?No, the IGV viewer has been limited to focus on your gene of interest. In the GridView, click a gene and the IGV viewer is updated automatically and centered onthat gene.

5. I’ve noticed that occasionally, the "Expected coverage" track for an amplicondoes not appear to contain information. Why is that?All amplicons in the design contain reads that are visualized in the "Expectedcoverage" track. If reads are not present, they are highlighted in the "Missedregions (if any)" track. It can happen that, if the number of reads covering anamplicon is relatively small in comparison to neighboring amplicons, the"Expected coverage" track appears empty. However, if you change the scale to alower value, you are then able to visualize the lower number of reads.

6. Why do some amplicons have very few reads in the "Expected coverage" track,versus others that have lots of reads?To achieve the most coverage (sensitivity), there is a sacrifice on specificity. So insome instances primers can either bind less tightly, or bind off-target, therebyreducing the number of amplicon reads at the desired region.



Spike-in Panels

1. What are Spike-in Panels?Spike-in Panels are high concentration Made-to-Order Panels that are used toextend the target range to be sequenced to include genes not available as On-Demand genes. Select the "Learn more" link for more information.

2. What is the benefit of a Spike-in Panel?Because the number of genes available as On-Demand genes is limited, a Spike-in Panel enables a user to sequence all the targets initially wanted in a singletarget amplification reaction.

3. What are the limitations of a Spike-in Panel?The limitations of Spike-in Panels involve the number of genes that can beincluded, and possible reduction in current expected coverage performance. Thesize of a compatible Spike-in Panel is limited to 123 amplicons per pool, for atotal of 246 amplicons. Any designs exceeding this limit will not be compatible.

4. How are Spike-in Panels different from Ion AmpliSeq™ On-Demand Panels?Spike-in Panels follow our Made-to-Order process and are synthesized de novofor every order, and the number of reactions is typically large (between 750 to3,000 reactions). On the other hand, On-Demand Panels have been optimized,premanufactured, tested and verified, and are available in small reaction numberbatches. On-Demand Panels also contain data that can be visualized in the IGVview that is available in the Grid view.



1. When should I use Ion AmpliSeq™ HD and not "regular" Ion AmpliSeq™?It depends on the application. If you have an application that requires ultra-highsensitivity, then Ion AmpliSeq™ HD is recommended. If you have applicationsthat require high multiplexing, then regular Ion AmpliSeq™ is the preferredoption.

2. What is the maximum number of amplicons per pool that are supported by IonAmpliSeq™ HD?Ion AmpliSeq™ HD currently supports up to 5,000 amplicons per pool with a 2-pool maximum of 10,000 amplicons total. This limit is enforced in AmpliSeq.com.

3. How many reactions worth of material can I expect in my order of an IonAmpliSeq™ HD panel?The approximate number of reactions worth of material provided for IonAmpliSeq™ HD panels is 3,000 reactions.

4. Designs are available only for cfDNA (75 to 100 bp amplicons) and FFPE (125to 175 bp amplicons), what about germline applications (125 to 275 bp and 125to 375 bp amplicons)?Germline applications may be considered in the future, depending on therelevance of use of Ion AmpliSeq™ HD technology.

5. What are the variant types supported by Ion AmpliSeq™ HD?The current supported variant types are SNVs, small INDELs, fusions from apredesigned list, and CNV through design best practices available in AmpliSeq.com.

6. What happens if my desired fusion is not available in AmpliSeq.com?In this case, a request to the AmpliSeq Custom Services team should besubmitted. Contact your local sales or support representative to learn more.

7. Does the AmpliSeq Custom Services team support specialty designs for IonAmpliSeq™ HD?Yes, the AmpliSeq Custom Services team is supporting specialty designs for IonAmpliSeq™ HD. Submission of specialty Ion AmpliSeq™ HD designs to theAmpliSeq Custom Services team should follow the same path for Ion AmpliSeq™

design requests, which involves contacting your sales or field support contactsfor help.

8. Only gene expression controls are available when creating a fusion design,what about custom designs for gene expression assays, are these supported byIon AmpliSeq™ HD?Automated gene expression designs are not currently supported in AmpliSeq.com. However, custom gene expression designs can be created by theAmpliSeq Custom Services team. Contact your local sales or supportrepresentative to learn more.

9. The new Oncomine cf PanCancer panel contains DNA and RNA targets in 1-pool, can you create a similar design with Ion AmpliSeq™ HD?Yes, but only through our AmpliSeq Custom Services team. Designs that arecreated in AmpliSeq.com are limited to 1-pool for DNA hotspots, 1-pool forRNA fusions, and 2-pools for DNA gene designs.

10. Why are the FWD and REV primers kept in separate pools?The primers used in Ion AmpliSeq™ HD are more complex than for regular IonAmpliSeq™, so it has been recommended to keep them in separate pools forstorage and long term stability. FWD and REV primers should only be mixed at

Ion AmpliSeq™ HDCustom panelsFAQs








the time the libraries are created. For more information, see the Ion AmpliSeq™

HD Library Kit User Guide (Pub. No. MAN0017392).

Related Ion sequencing products

This table provides links that take you to Thermo Fisher Scientific product descriptionpages.

Product description page link Description

Ion AmpliSeq™ Library Kit 2.0 This kit enables Ion AmpliSeq™ DNA and RNA librarypreparation from targeted DNA panels for any genome.

Ion AmpliSeq™ Library Kit Plus This kit enables Ion AmpliSeq™ DNA and RNA librarypreparation from targeted DNA panels for any genome.Recommended for manual library preparation for IonAmpliSeq™ On‑Demand Panels.

Ion AmpliSeq™ HD Library Kit This kit enables Ion AmpliSeq™ HD DNA and RNA dual-barcoded library preparation from targeted DNA and RNApanels.

Ion AmpliSeq™ Sample ID Panel This kit is a versatile, cost effective, and easy-to-usehuman SNP genotyping panel comprising 9 speciallydesigned primer pairs that can be added to the multiplexPCR reaction to generate a unique ID during post-sequencing analysis of research samples.

Ion Library Equalizer™ Kit This kit is a simple and seamless bead-based solutionreplacing the need for library quantification and librarydilutions for library normalization as required for anynext generation sequencing workflow. This kit helpsreduce labor, reagent costs, and further simplifies the Ionsemiconductor sequencing workflow for high sample-throughput or barcoded projects, as well as single or lowsample number projects.

Ion Xpress™ Barcode Adapters 1–16 Kit This kit provides a set of 16 unique barcode adaptorsspecifically designed and verified for optimalperformance with the Ion PGM™, Ion Proton™, Ion S5™/IonS5™ XL, and Ion GeneStudio™ S5 Series sequencers.When used in combination with the Ion AmpliSeq™

Library Kit 2.0 or the Ion AmpliSeq™ Library Kit Plus, thiskit enables users to pool up to 16 libraries beforetemplate preparation, and then conduct multiplexedsequencing analysis, simplifying the Ion sequencingworkflow for a wide range of applications. Use of this kitwith other Ion Xpress™ Barcode Adapters kits allowspooling of up to 96 amplicon or fragment libraries.

IonCode™ Barcode Adapters 1–384 Kit This kit provides 384 different pre-mixed adapters in aconvenient 96-well plate format.

Ion AmpliSeq™ HD Dual Barcode Kit 1–24 This kit provides 24 dual barcode primers for IonAmpliSeq™ HD library preparation.

Appendix A Supplemental informationRelated Ion sequencing products A


https://www.thermofisher.com/order/catalog/product/4475345?ICID=search-4475345

https://www.thermofisher.com/order/catalog/product/A38875?SID=srch-srp-A38875





https://www.thermofisher.com/order/catalog/product/A29751?ICID=search-A29751


Product description page link Description

Ion AmpliSeq™ Kit for Chef DL8 This kit enables automated Ion AmpliSeq™ DNA and RNAlibrary preparation of up to 8 libraries per run on theIon Chef™ System.

Ion PGM™ Hi‑Q™ View Chef Kit

Ion PI™ Hi‑Q™ Chef Kit

Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef

Ion 540™ Kit – Chef

Ion 550™ Kit − Chef

Ion Chef™ template and sequencing kits enable robustand automated template preparation, chip loading, andsequencing of up to 400-base libraries on the Ion PGM™,Ion Proton™, Ion S5™, Ion S5™ XL, and Ion GeneStudio™ S5Series sequencers.

Ion 314™ Chip Kit v2 BC



Ion PI™ Chip Kit v3

Ion 510™ Chip Kit

Ion 520™ Chip Kit

Ion 530™ Chip Kit

Ion 540™ Chip Kit

Ion 550™ Chip Kit

These kits contain chips for sequencing runs on the IonPGM™, Ion Proton™, Ion S5™, Ion S5™ XL, and IonGeneStudio™ S5 Series sequencers. The chipselectronically detect polymerase-driven baseincorporation without the use of fluorescence. Byeliminating the use of an optical detection system, thisadvance in next-generation sequencing technology allowsfor rapid sequencing times of as little as 2.5 hours for 200bp sequencing, with little hands-on time from sample tosequencing data.

Helpful tools

This table provides links that take you to third-party tools that are helpful whendesigning panels.

Link to tools Description

UCSC Table Browser Use this program to retrieve the dataassociated with a track in text format, tocalculate intersections between tracks, andto retrieve DNA sequence covered by atrack.

UCSC Genome Browser This site contains the reference sequenceand working draft assemblies for a largecollection of genomes.

Integrative Genomics Viewer (IGV) The Integrative Genomics Viewer (IGV) is ahigh-performance visualization tool forinteractive exploration of large, integratedgenomic datasets. It supports a wide varietyof data types, including array-based andnext-generation sequence data, andgenomic annotations.

Appendix A Supplemental informationHelpful toolsA


https://www.thermofisher.com/order/catalog/product/A29024?ICID=search-product

https://www.thermofisher.com/order/catalog/product/A29902?ICID=search-product





https://www.thermofisher.com/order/catalog/product/4488144?SID=srch-srp-4488144

https://www.thermofisher.com/order/catalog/product/4488145?SID=srch-srp-4488145

https://www.thermofisher.com/order/catalog/product/4488146?ICID=search-product









http://software.broadinstitute.org/software/igv/

Advanced features and tools

Ion AmpliSeq™ Designer links the output designed BED file directly to the UCSCGenome Browser automatically configured with the dbSNP and RepeatMaskerannotation tracks. Although this allows you to determine if the regions were filteredbased on either repeat regions or SNPs under the 3' primer, the Ion AmpliSeq™

Designer also filters for other reasons. This module offers some suggestions for usingother bioinformatics tools to determine the quality of the design and for researchpurposes.

One common reason that Ion AmpliSeq™ Designer filters a region would be that it is anonunique sequence and would hybridize in other areas of the genome and thus bedeleterious to your reaction. BLAST, the Basic Local Alignment Search Tool, findsregions of local similarity between sequences.

1. Using the UCSC Genome Browser, enter the region that has been filtered intothe results of your design.

Minimize off-targethybridization

Appendix A Supplemental informationAdvanced features and tools A



2. Copy the sequence, and run a BLAST search on this sequence.

3. Select Human, running a nucleotide query.

If the search returns multiple results for that query, Ion AmpliSeq™ Designer hasfiltered this to minimize off-target hybridization.

Appendix A Supplemental informationAdvanced features and toolsA


Ion AmpliSeq™ Designer filters regions that have either very high or very low GCcontent.

1. Using the UCSC Table Browser, select the Mapping and Sequencing Tracksgroup option and the GC Percent track option.

2. In the Enter region definition dialog box, you can enter or paste a regiondefinition to search for a specific region, or upload your designed BED file to theUCSC Table Browser.The output appears in a 5-basepair sliding window where you can determineregions that are below 20% or above 80% in GC content.

Determine GCcontent

Appendix A Supplemental informationAdvanced features and tools A



You can use the coverageAnalysis plugin in Torrent Suite™ Software to determinecoverage uniformity of your sequence run. For further information on using thecoverageAnalysis plugin, see the Torrent Suite™ Software Help.

AddcoverageAnalysisplugin

Appendix A Supplemental informationAdvanced features and toolsA


Documentation and support

Related documentation

Document Pub No.

Ion AmpliSeq™ Library Kit 2.0 User Guide MAN0006735

Ion AmpliSeq™ Library Kit Plus User Guide MAN0017003

Ion AmpliSeq™ HD Library Kit User Guide MAN0017392

Oncomine™ tumor specific panels User Guide MAN0018518

Qubit™ dsDNA HS Assay Kits User Guide MAN0002326

Demonstrated Protocol: Sample Quantification for Ion AmpliSeq™ Library PreparationUsing the TaqMan™ RNAse P Detection Reagents Kit MAN0007732

Ion Library TaqMan™ Quantitation Kit User Guide MAN0015802


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 forthin the Life Technologies' General Terms and Conditions of Sale at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you haveany questions, please contact Life Technologies at www.thermofisher.com/support.

Documentation and supportCustomer and technical support


http://thermofisher.com/support

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Ion AmpliSeq Designer: Getting Started · reactions) for library preparation on the Ion Chef ... Ion AmpliSeq™ Designer: Getting Started User Guide 7. 1 2 3 7-11 1 Start a design