ion ampliseq designer: getting started - amazon s3 · 2017-06-02 · in ion ampliseq™ designer,...

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

Ion AmpliSeq™ Designer: Getting StartedUSER GUIDE

Publication Number MAN0010907

Revision E.0

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

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

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

Revision history: MAN0010907

Revision Date DescriptionE.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.

©2017 Thermo Fisher Scientific Inc. All rights reserved.

Contents

■ CHAPTER 1 Getting started with Ion AmpliSeq™ Designer . . . . . . . . . . . 5

Browse Ion AmpliSeq™ Ready-to-Use and Community Panels . . . . . . . . . . . . . . . . . . . . . . . . . 6

Navigation bar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Chip Calculator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

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

Copy existing amplicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Known limitations of Copy amplicons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Basic and biological filtering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Filtering of repeat regions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18Biological filtering ‑ GC content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Repeat regions and RepeatMasker filtering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

■ CHAPTER 2 Start an Ion AmpliSeq™ On‑Demand Panel design . . . . 20

Start a new On-Demand design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

■ CHAPTER 3 Start an Ion AmpliSeq™ Made-to-OrderPanel design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Start a new DNA or RNA panel design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Start an RNA gene fusion design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Add gene expression assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

Ion AmpliSeq™ Designer: Getting Started User Guide 3

■ APPENDIX A Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

■ APPENDIX B Supplemental information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Padding coordinates or create a BED file from list of variants . . . . . . . . . . . . . . . . . . . . . . . . 41Create a BED file for input into Ion AmpliSeq™ Designer . . . . . . . . . . . . . . . . . . . . . . . . 41Work from a list of COSMIC IDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Start from a list of dbSNP target identifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42Use the UCSC Genome Browser to create a BED file with Padded Exons . . . . . . . . . . 44

Reference FASTA sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Known polymorphism BED file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Pipeline details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46The design pipeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Tiling algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Pooling algorithm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Advantages of pooling and tiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Hotspots pipeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Frequently asked questions (FAQs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50General Ion AmpliSeq™ Designer FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50Ion AmpliSeq™ On‑Demand Panels FAQs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

Related Ion sequencing products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Helpful tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Advanced features and tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58Minimize off-target hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58Determine GC content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60Add coverageAnalysis Plugin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

■ Documentation and support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Contents

4 Ion AmpliSeq™ Designer: Getting Started User Guide

Getting started with Ion AmpliSeq™

Designer

Welcome to Ion AmpliSeq™ Designer! This chapter will describe some procedures andtools used in Ion AmpliSeq™ Designer, and will show how to browse predesignedReady-to-Use and Community Panels. Chapters 2 and 3 will cover how to design andorder Ion AmpliSeq™ On-Demand and Made-to-Order Panels.

Before you begin a panel design or browse predesigned panels, sign in at AmpliSeq.com with your Thermo Fisher Scientific user name and password. Thehome page shows the options available for designing and ordering Ion AmpliSeq™

panels, and shows the latest product news. Three design and ordering options arenow available:

• Ready-to-Use Panels: predesigned DNA and RNA panels for germline or somaticanalysis, ordered in small reaction packs.

• On-Demand Panels: DNA panels of optimized amplicons for germline analysis.Configurable to a specific human disease area, and ordered in small reactionpacks.

• Made-to-Order Panels: DNA and RNA designs for germline or somatic analysisof any genome, ordered in large reaction packs. Made-to-Order Panels wereformerly known as Custom Panels.

1


https://www.ampliseq.com

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

1. Sign in to Ion AmpliSeq™ Designer with your Thermo Fisher Scientific log ininformation.

2. To browse pre-designed Ion AmpliSeq™ panels, click DNA or RNA in the Ready-to-Use Panels section.

A table displaying research panels and their descriptions appears.

Chapter 1 Getting started with Ion AmpliSeq™ DesignerBrowse Ion AmpliSeq™ Ready-to-Use and Community Panels1


3. Select an area of interest, such as Cancer Research, if desired, to filter the results.To see multiple categories, select all research areas of interest from the list.

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

Chapter 1 Getting started with Ion AmpliSeq™ DesignerBrowse Ion AmpliSeq™ Ready-to-Use and Community Panels 1


You can also browse pre-designed Community Panels in the same research areasby clicking the Pre-designed by our Community button in the Made-to-OrderPanels section on the right side of the home screen.

Navigation bar

The navigation bar provides easy access to areas of interest.

4321

1 Home – Takes you to starting point for creating a custom panel design.2 My Designs – Allows you to see the designs you have already created.3 Notifications – Provides updates of new features and messages from ampliseq.com.4 Help – Takes you to the customer help page.

Chapter 1 Getting started with Ion AmpliSeq™ DesignerNavigation bar1


Chip Calculator

The Chip Calculator provides recommendations for maximum sample library loadingper chip for a given panel, chip, and coverage (500X for somatic samples, and 30X forgermline samples). You can use the calculator as a guide for library loading for Ready-to-Use panels, and to aid in the design of a Made-to-Order panel. Access the ChipCalculator by clicking the Review panel button of a Ready-to-Use or Communitypanel, and in the Results ready page that is returned after you submit a panel design.

As you proceed down the information list, the dropdown options become application-specific. Also note that instrument selection changes the Chip options in the lowertable.

Chapter 1 Getting started with Ion AmpliSeq™ DesignerChip Calculator 1


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

Chapter 1 Getting started with Ion AmpliSeq™ DesignerChip Calculator1


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 your own. The following stepsdescribe how to upload a custom reference.

1. From the navigation bar, select Pre-loaded Genomes.

The Design References screenappears and Public Genomes aredisplayed. If you have previouslyuploaded custom references, clickthe Custom References tab to viewthem.

Note: You cannot select a publicgenome at this point. Publicgenomes are the available choiceswhen you start a new design. Here,the list of public genomes is forinformational purposes only.

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

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


3. Fill in the required information on the Add a custom reference screen, then clickSelect file under Reference sequences (FASTA file). Navigate to the location ofthe FASTA file on your drive, then select it.

1

2

3

4

5

6

7

1 Reference Name – Must be composed of US-ASCII letters, numbers, and spaces,between 3 and 32 characters in length.

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


2 Associated organism for primer specificity check – Click to view the dropdown menucontaining list of organisms. If your data are associated with one of our supportedorganisms, providing this information can improve primer specificity to your customreference by favoring primers with few optimal binding sites in the consensussequence. Primer specificity check refers to the process of identifying potentialprimer mispriming events. Primers with high number of potential mispriming eventsare avoided in our designs.

3 Reference source (Recommended) – Name of the database/source of the DNAsequence.

4 Reference description – Add any notes regarding the custom reference sequence.5 Reference sequences – You can either upload a FASTA file (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 – composed of 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 other variation).Ion AmpliSeq™ Designer minimizes primer overlap with these regions. This file isoptional. See the Appendix for specifications on creating and formatting BED files foruploading.

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

5. After the upload progress bars complete, click Save. When the upload procedurefinishes, select the Custom References tab to view your uploaded reference.



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

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


7. Click the Edit button at the upper right to edit the following: Reference name,Reference source, and Reference description.

Note: Updates to these textual identifiers are made throughout the entire site.

Note: You cannot change the uploaded files (genomic data) as they arepermanently associated with this assigned custom reference genome. To makechanges, delete and re-upload your edited files using the Add reference button.Click Delete to remove the reference from the list of active custom references.This action does not affect existing designs — associated custom references arestill downloadable.



8. When building your custom panel, click Custom Reference, then select yourcustom reference from the dropdown list.

Copy existing amplicons

Amplicons from one or more Ion AmpliSeq™ Ready-to-Use Panels, CommunityPanels, and/or previous Made-to-Order designs can be copied and resubmitted intothe pipeline to generate new designs, without needing to calculate the position of theamplicon.

1. Create a new draft design from the home page, then determine the source of theamplicons you want to copy from (Ready-to-Use Panels, Community Panels,and/or previous designs). To copy amplicons from Ready-to-Use and/orCommunity Panels:

a. Click the Browse by DNA or RNA button, or the Pre-designed by ourCommunity button, then select your panel type.

b. Select the panel that you are interested in subsetting from, then click Reviewpanel.

Chapter 1 Getting started with Ion AmpliSeq™ DesignerCopy existing amplicons1


2. Check the box to select your gene of interest, then click Copy amplicons to copyall amplicons to your new design:

3. A dialog appears asking you to confirm which target rows you want to copy.Make your selection, then click Copy amplicons to another design.

a. Copy the amplicons to a pre-existing design by selecting one from thedropdown, or enter a new design name.

Note: If you are copying amplicons into a pre-existing design, ampliconsmust be of the same genome or in draft form only.

4. To view the list of amplicons and/or select specific ones to copy, click the generow and the specific region of interest to view the Amplicon ID(s).

a. Highlight and copy the Amplicon ID for the amplicon(s) you want to copy,then paste them into the utility bar to add them manually to your newdesign:

Note: Follow these same steps to manually copy and add specific AmpliconIDs from previous designs and/or additional panels. You can also manuallyenter predesigned genes(s)/region(s) or upload a list of amplicons/geneusing the other utilities.

5. After you compile a list of targets for which you want to create a new design,click Submit targets, then confirm your submission, as before.

Note: Subset designs can be combinations of pre-designed regions andamplicons from custom designs mixed and matched with amplicons from othercustom designs or Ready-to-Use and Community Panels.

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


• 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 File"method. Amplicons can only be copied using the Copy amplicons button anddialog in the UI.

• The following DNA panels are not compatible for use with the Copy ampliconsfunction:

– A25643 - HID-Ion AmpliSeq™ Identity Panel– A25642 - HID-Ion AmpliSeq™ Ancestry Panel– Ion AmpliSeq™ Exome Panel– Ion AmpliSeq™ Exome Panel (Hi-Q)– Ion AmpliSeq™ Colon and Lung Cancer Panel

• 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.

Basic and biological filtering

The human genome is heavily populated with repeat regions that make designingprimers difficult, a well known challenge in PCR design. The Ion AmpliSeq™ Designerpipeline has been developed to deliver the most robust set of amplicons it cangenerate. The pipeline specifically excludes amplicons that are placed in repeatelements or other hypervariable regions to generate the best possible outcome foractual amplicon coverage when used in a reaction.

A focus in our Research & Development department is to better understand theproperties of repeat regions to allow primer placement in these regions to achievehigher target design rate while maintaining coverage uniformity and on-target rates.

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

Designer pipeline to evaluate repeat elements is the RepeatMasker. RepeatMasker is aprogram that screens DNA sequences for interspersed repeats and low complexityDNA sequences, and is an annotation track that is available through the UCSCGenome Browser. One of the new feature upgrades in v1.2 of the Ion AmpliSeq™

Custom Designer links directly to the browser and offers the user the visualrepresentation to distinguish between three BED files as custom annotation tracks.

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

• 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 browser)

Known limitationsof Copy amplicons

Filtering of repeatregions

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


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 611amplicons.

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

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

Biologicalfiltering ‑ GCcontent

Repeat regionsandRepeatMaskerfiltering

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


Start an Ion AmpliSeq™ On‑DemandPanel design

Ion AmpliSeq™ On-Demand Panels represent a new way to design panels for use ininherited 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.Presently, Ion AmpliSeq™ On-Demand Panels are limited to 300 genes and/or 9,000amplicons. The gene designs in the On-Demand catalog have been optimized for highperformance. You can add genes that are not in the On-Demand catalog to your panelas a Spike-in Panel.

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

Start a new On-Demand design

1. Sign in to Ion AmpliSeq™ Designer with your Thermo Fisher Scientific logininformation. If you are already signed in but not on the home page, click theHome icon in the navigation bar to return to the home page, then click theDisease Research Areas button.

2


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

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

Chapter 2 Start an Ion AmpliSeq™ On‑Demand Panel designStart a new On-Demand design 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 300, and the total number of amplicons cannot exceed 9,000. 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 by keyword, research area/gene symbol field inthe upper right corner to add Disease Research Areas to your design.

4. When you have completed your selections, enter a name for your panel design,then click Proceed.

5. On the panel order page, you can:• add additional genes to the panel design, either individually or as an

uploaded list• remove a gene from the design by deselecting its checkbox• use the IGV to view the exon structure, amplicon coverage, missed regions,

and general information for eachgene• create a Spike-in Panel by selecting Disease Research Area genes

Chapter 2 Start an Ion AmpliSeq™ On‑Demand Panel designStart a new On-Demand design2


• order the panel• copy or clone an ordered and locked panel to make further changes

6

1 432

5

7

8

9

1 Order — click when you have finished your panel design2 Clone Panel — copy the panel and add or delete genes3 Download Results — download results files after a design has been ordered, or a Spike-in Panel has been ordered4 Export Targets — generate an Excel .csv file of the target list5 Upload File — upload a .csv file containing a list of genes to include in a panel6 Add Gene — enter gene symbol to upload genes to panel design individually7 Grid/Table — toggle between the grid (shown) and table views of target genes8 Sort By — sort target list by score, a ranking of the relationship between the disease and gene, or alphabetically9 Genes available as On-Demand or not available as On-Demand (Spike-in Panel)



Select a gene to view its genomic and amplicon information in the IGV section.

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

Chapter 2 Start an Ion AmpliSeq™ On‑Demand Panel designStart a new On-Demand design2


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

IMPORTANT!· Spike-in Panel designs must contain at most 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

only be edited if it is cloned.

8. When you have completed your design, click Order.



Start an Ion AmpliSeq™ Made-to-Order Panel design

Start a new DNA or RNA panel design

If you are using one of the Ion AmpliSeq™ standard genome references, starting a newdesign is a simple process.

Sign in to Ion AmpliSeq™ Designer with your Thermo Fisher Scientific logininformation. If you are already signed in but not on the home page, click the Homeicon in the navigation bar to return to the home page, then click Genes, Regions, orAmplicons.

3


1. When the Start a new design page opens, enter a Design Name and, optionally,details.

2. Select your Application type. Select DNA Gene designs (multi-pool), DNAHotspot designs (single-pool), RNA Gene Expression designs (single-pool), orRNA Gene Fusion designs (multi-pool). Clicking the application type filters thecompatible genomes shown.

Note: Custom reference genomes are currently only compatible with DNAdesigns. RNA Gene designs are only compatible with the human genome.

3. Select a genome to use as reference. For custom references, click CustomReference.

4. Click Next: Add Targets to proceed.

Chapter 3 Start an Ion AmpliSeq™ Made-to-Order Panel designStart a new DNA or RNA panel design 3


5. Three options for adding targets are available: Add Gene/Region, AddAmplicons by ID, and Upload File.

Option Description

Add Gene/Region Allows various manual options:

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

2. Start typing the gene symbol or region.

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

4. When finished, click Submit targets.

Add Amplicon byID

Allows you to enter amplicon IDs assigned to specific genomiccoordinates.

Upload File Allows you to upload genomic coordinates of several targets at atime, via a CSV or BED file (select from the "Type" dropdown).

Ion AmpliSeq™ Designer uploads the targets, checks them, and verifies regions.

6. For any highlighted erroneous target(s), either correct the coordinates inside thetable, or remove them by checking their checkbox(es), then clicking the Deletebutton.

7. (Optional) Click Export targets to download your targets into a CSV file.

Chapter 3 Start an Ion AmpliSeq™ Made-to-Order Panel designStart a new DNA or RNA panel design3


8. To submit your panel, click Submit targets. Two designs can be submitted at atime.

9. Confirm your submission.

When confirmed, you see a confirmation message and receive an emailconfirming the design submission.

10. When the Assay Design results are ready, you receive an email instructing you toreview the results in Ion AmpliSeq™ Designer. Click the View results linkprovided in the email 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 Start an Ion AmpliSeq™ Made-to-Order Panel designStart a new DNA or RNA panel design 3


https://www.ampliseq.com

11. Use the following fields to review your order:

8

1 2 3 4

9765 10 11

1 Switch design—Click the dropdown to change the design in view.2 Edit—Allows you to edit the Design Name and Details only.3 Copy Targets—Allows you to copy your design to modify it.4 Start a new design—Starts a new design.5 Add to cart—When this button is green you can click it to add the highlighted design to your cart.

Note: If there are not sufficient amplicons (at least 12), the button is grayed out and a message appears.6 Chip Calculator— 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.

Note: Sharing your design also makes your custom reference available for review and downloading by anyone to whomyou provide the 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.

Chapter 3 Start an Ion AmpliSeq™ Made-to-Order Panel designStart a new DNA or RNA panel design3


Start an RNA gene fusion design

To make RNA Gene Fusion panels with a combination of Gene Fusion and GeneExpression Assays (GEX), follow the steps below.

IMPORTANT! For successful panels, you must have at least 12 GEX assays per panel.We provide 12 default GEX assays for each panel that you can accept or replace withtargets of your choice. For panels requiring two pools, the GEX assays are splitbetween the two pools.

1. If you are already on AmpliSeq.com working in another application, click theHome icon in the navigation bar to return to the main page.

2. Click the Genes, Regions or Amplicons button in the Made-to-Order Panelssection, then enter a Design Name and, optionally, details.

Chapter 3 Start an Ion AmpliSeq™ Made-to-Order Panel designStart an RNA gene fusion design 3


3. Select RNA Gene Fusion designs (multi-pool) button, in application type.

Note: Human (RefSeq/Ensembl) is the only genome that is permitted with thispanel type.

Chapter 3 Start an Ion AmpliSeq™ Made-to-Order Panel designStart an RNA gene fusion design3


4. Click Next: Add Targets.At the top of the Review Draft Designs screen, you can Add Fusions by SymbolPair or upload them from a file.Click "Input Specifications" link for details. At the bottom of the screen, you seetwo tabs, Fusions to add gene fusion targets, and Genes to add gene expressiontargets. These tabs represent the two stages that are mentioned in theintroduction for this section. The Fusions tab is selected by default.

5. On the Add Fusion tab of the upper section, add your targets.a. In the Symbol Pair text box, start typing the gene symbol that you would

like to add targets for, and the available fusions partners are displayed.



b. Select the desired gene pair.

c. Add the fusions by clicking the checkbox to the left of each target, then clickAdd fusion(s).

6. Repeat step 4 a-c to add additional targets.Ion AmpliSeq™ Designer uploads the targets and checks them. After completion,a status message appears at the top of the screen: either "Target savedsuccessfully" in green text, or "# duplicate fusions ignored" in red text.



Now add your gene expression assays. Every pool is required to have 12 geneexpression assays, and we have pre-selected them to get you started. To pick yourown gene expression assays, delete the proposed ones and add your own geneexpression targets by gene symbol or RefSeq transcript accession.

1. Click the Genes 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 the InputSpecifications link in the software user interface for more details and a CSVtemplate for creating your own list of genes or RefSeq Accession numbers.

Note: If you would like to restore the pre-populated gene expression assays,click Restore pre-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. Two designs can be submitted at a time.

5. Confirm your submission.

Note: You can submit a second submission, if needed.

Acknowledgement of the submission appears at the top of the page, and is alsosent to you via email.

6. When your fusion results are ready, you receive an email instructing you toreview the results in Ion AmpliSeq™ Designer.

Note: You may need to check your spam folder and move the email to yourInbox to enable its links.

Add geneexpression assays



7. Click the link provided in the email, or go to the Ion AmpliSeq™ Designerwebsite and navigate to My Designs4RNA.

8. Click the name of your design in the Design column.



9. Review results on the Fusions tab, then make changes if needed by clickingCopy Targets and submitting a new design with your changes.

5

1 2 3 4

76 8 9

1 Switch design – Click the dropdown menu to change the design in view.2 Edit – lets you edit the Design Name and Details only.3 Copy Targets – lets you copy your design to modify it.4 Add design – Starts 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. Review gene assay results on the Genes tab.



11. Click the Gene link to view compatible transcripts.



Troubleshooting

For complete Ion AmpliSeq™ library troubleshooting, see the Ion AmpliSeq™ Library Kit2.0 User Guide (Pub. No. MAN0006735), or the Ion AmpliSeq™ Library Kit Plus UserGuide (Pub. No. MAN0017003), available at thermofisher.com. The following tablecan help determine probable cause and recommended action for several performanceobservations.

Observation Possible cause Recommended action

Short amplicons are under-represented Purification was poor. Vortex AMPure™ XP Reagentthoroughly before use, and besure to dispense the fullvolume.

100% ethanol is difficult topipet accurately; it isessential to pre-wet pipettetips.

In post-ligation librarypurification, increaseAMPure™ XP Reagent volumefrom 45 μL (1.5X) to 50 μL(1.7X).

Long amplicons are under-represented (shortlibrary reads)

Sample DNA or RNA wasdegraded.

Use an FFPE assay design fordegraded or low qualitysamples.

PCR was inefficient. Double the anneal and extendtime.

Too few nucleotide flows wereused.

Use an appropriate number offlows to sequence throughamplicons.

Sample was over-treated withFuPa Reagent.

Add no more than 2 µL FuPaReagent per 20 µL targetamplification reaction.

Keep the plate on ice duringFuPa Reagent addition, thentransfer to a preheatedthermal cycler immediately.

Denaturation temperaturewas too high.

Use a 97°C enzymeactivation/denaturationtemperature instead of 99°Cin target amplificationreactions.

A


http://www.thermofisher.com

Observation Possible cause Recommended action

AT-rich amplicons are under-represented

Example of loss of AT-rich amplicons. Within theCoverage Analysis Plugin, ampliconrepresentation is plotted by GC content for an IonAmpliSeq™ Panel. Amplicons with 23-50% GCshow an excess failure rate (less than 20% of themean read depth).

Target amplification wasinefficient.

Double the anneal/extendtime in the targetamplification reaction.

Decrease the anneal/extendtemperature of the targetamplification reaction from60°C to 58°C.

Digested amplicons weredenatured.

Do not exceed 60°C duringthe amplicon digestion step.

Digestion was inefficient. Increase amplicon digestiontimes to 20 minutes for eachstep.

GC-rich amplicons are under-represented

Example of loss of GC-rich amplicons. Within theCoverage Analysis Plugin, ampliconrepresentation is plotted by GC content for an IonAmpliSeq™ Panel. Amplicons with 60-80% GCshow an excess failure rate (less than 20% of themean read depth).

Denaturation was inadequateduring target amplification.

Use a calibrated thermalcycler.

Target amplification wasinefficient.

Increase the anneal/extendtemperature of the targetamplification reaction from60°C to 62°C for the first twocycles of the targetamplification reaction

Library amplification wasinefficient.

Do not amplify the library (notrequired for qPCRquantification).

Sample was over-treated withFuPa Reagent.

Add no more than 2 µL FuPaReagent per 20 µL targetamplification reaction.

Keep the plate on ice duringFuPa Reagent addition, thentransfer to thermal cyclerimmediately.

Appendix A TroubleshootingStart an RNA gene fusion designA


Supplemental information

■ Padding coordinates or create a BED file from list of variants . . . . . . . . . . . . . . . 41

■ Reference FASTA sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

■ Known polymorphism BED file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

■ Pipeline details . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

■ Hotspots pipeline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

■ Frequently asked questions (FAQs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

■ Related Ion sequencing products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

■ Helpful tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

■ Advanced features and tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

Padding coordinates or create a BED file from list of variants

You can generate a custom BED file with your target regions of interest from the UCSC Table Browser.

To create a BED-formatted file starting from a list of either COSMIC mutations ordbSNP identifiers, use the UCSC Table Browser of the UCSC Genome Browser. Makesure to select genome and assembly as Human, (GRCh37/hg19).

1. Select All Tracks in the group drop-down list, and COSMIC for the track.

2. Choose the COSMIC option in the table list.

B

Create a BED filefor input into IonAmpliSeq™

Designer

Work from a list ofCOSMIC IDs


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

https://genome.ucsc.edu/

1. Select the Variation and Repeats group in the drop-down menu. The track andtable sections are then populated with available SNP databases from dbSNP.

2. Choose the genome radio button for the region selection. Click on either thepaste list or upload list button next to identifiers.

3. Either upload a text file listing your COSMIC or dbSNP ids or simply paste in thelist and click submit. For the output format, select BED - browser extensible data.

Start from a list ofdbSNP targetidentifiers

Appendix B Supplemental informationPadding coordinates or create a BED file from list of variantsB


4. After clicking on the get output button, the Output as BED screen allows forpadding with Whole Gene, Upstream or Downstream.

The BED file will be similar to the following format:

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

Appendix B Supplemental informationPadding coordinates or create a BED file from list of variants B


Using the Table Browser, select the following options:• Group: Genes and Gene Prediction Tracks• Track: RefSeq Genes• Table: refGene• Output format: BED - browser extensible data

1. Upload or paste a list of gene identifiers. When you click submit, you will bedirected to the Output refGene as BED page.

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

3. Additionally, select to create the BED file to include 5' or 3' UTR Exons or CDS.

Use the UCSCGenome Browserto create a BEDfile with PaddedExons

Appendix B Supplemental informationPadding coordinates or create a BED file from list of variantsB


Reference FASTA sequence

Uploading sequences

One or more reference sequences in FASTA format can be uploaded by:

a. Selecting a "plain text" or compressed file (in either ZIP or GZIP formats) containingthe sequence(s). The maximum file size allowed for upload is 1 GB afterdecompression.

b. Copying and pasting the sequences in the text area available after clicking on thelink "Enter FASTA data in a text area instead."

FASTA format

A sequence in FASTA format is expressed in 2 or more lines of text: The first line is anidentifying "header", the rest of the lines (one or more) represent the sequence itself.

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

• The sequence: The only characters accepted for representing a sequence are "A,""C," "G," "T," and "N" (lower case versions are also allowed for representing lowcomplexity regions). Although a sequence can be just one or multiple lines ofdifferent size after the header, it is traditional to use separate lines of 50 or 60characters 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 (i.e., SNPs, indels, or other variation).Ion AmpliSeq™ Designer will minimize primer overlap with these regions. This file isoptional. You may 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 will be 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.

Appendix B Supplemental informationReference FASTA sequence B


They must meet the following criteria:• chromStar t may be a value between 0 and length of the sequence specified by

chrom minus 1.• chromEnd must be greater than chromEnd.• chromEnd may have a maximum value of the length of the sequence specified by

chrom.• No region should overlap any other region in the file. Overlapping regions

should be merged by the customer into a single contiguous region.• The variant coordinates BED file must have no header (i.e., no "track" lines).

Example FASTA file (50 bases per line):

Sample variants of interest (highlighted in blue).

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.

Appendix B Supplemental informationPipeline detailsB


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

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 designpipeline

Tiling algorithm

Appendix B Supplemental informationPipeline details B


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:

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.

• 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 designs represent cost and handling advantages over panels that requiretwo or more pools of primers. Designs targeting small genomic regions such as SNPsand small indels are well suited to fit in a single pool because those regions can becovered 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.

Pooling algorithm

Advantages ofpooling and tiling

Appendix B Supplemental informationHotspots pipelineB


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.

Appendix B Supplemental informationHotspots pipeline B


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.

General IonAmpliSeq™

Designer FAQs

Appendix B Supplemental informationFrequently asked questions (FAQs)B


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 B Supplemental informationFrequently asked questions (FAQs) B


General Ion AmpliSeq™ On-Demand Panel design questions

1. Why can I order only 300 genes in an On-Demand panel?For this version of the software, we have set an ordering limit to 300 genes or9,000 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 300 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 a gene and a disease. It takes intoaccount both the strength and number of 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 evidencedemonstrating that 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) for newborns.

IGV Viewer

1. What is the "Expected coverage" track in the IGV viewer?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,pre-manufactured, tested and verified, and are available in small reactionnumber batches. On-Demand Panels also contain data that can be visualized inour integrated IGV viewer available in the Grid view.



Related Ion sequencing products

Link to product purchase page Description

Ion AmpliSeq™ Library Kit 2.0 Ion AmpliSeq™ DNA and RNA librarypreparation from targeted DNA panels forany genome.

Ion AmpliSeq™ Library Kit Plus Ion AmpliSeq™ DNA and RNA librarypreparation from targeted DNA panels forany genome. Recommended for manuallibrary preparation for Ion AmpliSeq™

On‑Demand Panels.

Ion AmpliSeq™ Sample ID Panel The Ion AmpliSeq™ Sample ID Panel is aversatile, cost effective, and easy-to-usehuman SNP genotyping panel comprising 9specially designed primer pairs that can beadded to the multiplex PCR reaction togenerate a unique ID during post-sequencing analysis of research samples.

Ion Library Equalizer™ Kit The Ion Library Equalizer™ Kit is a simpleand seamless bead-based solutionreplacing the need for library quantificationand library dilutions for librarynormalization as required for any nextgeneration sequencing workflow. The IonLibrary Equalizer™ Kit helps reduce labor,reagent costs, and further simplifies the Ionsemiconductor sequencing workflow forhigh sample-throughput or barcodedprojects, as well as single or low samplenumber projects.

Ion Xpress™ Barcode Adapters 1–16 Kit The Ion Xpress™ Barcode Adapters 1–16 Kitprovides a set of 16 unique barcodeadaptors specifically designed and verifiedfor optimal performance with the IonPGM™, Ion Proton™, and Ion S5™/Ion S5™ XLsequencers. When used in combination withthe Ion AmpliSeq™ Library Kit 2.0 or the IonAmpliSeq™ Library Kit Plus, this kit enablesusers to pool up to 16 libraries beforetemplate preparation, and then conductmultiplexed sequencing analysis,simplifying the Ion sequencing workflow fora wide range of applications. Use of this kitwith other Ion Xpress™ Barcode Adapterskits allows pooling of up to 96 amplicon orfragment libraries.

IonCode™ Barcode Adapters 1–384 Kit The IonCode™ Barcode Adapters 1–384 Kitprovides 384 different pre-mixed adaptersin a convenient 96-well plate format.

Appendix B Supplemental informationRelated Ion sequencing productsB


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




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

Link to product purchase page Description

Ion AmpliSeq™ Kit for Chef DL8 The Ion AmpliSeq™ Kit for Chef DL8 enablesautomated Ion AmpliSeq™ DNA and RNAlibrary preparation of up to 8 libraries perrun on the Ion Chef™ System.

Ion PGM™ Hi‑Q™ View Chef Kit

Ion PI™ Hi‑Q™ Chef Kit

Ion 520™ & Ion 530™ Kit – Chef

Ion 540™ Kit – Chef

Ion Chef™ template and sequencing kitsenable robust and automated templatepreparation, chip loading, and sequencingof up to 400-base libraries on the IonPGM™, Ion Proton™, Ion S5™, and IonS5™ XL sequencers.

Ion 318™ Chip v2 BC

Ion PI™ Chip Kit v3

Ion 530™ Chip Kit

Ion 540™ Chip Kit

Ion Chip kits contain chips for sequencingruns on the Ion PGM™, Ion Proton™, IonS5™, and Ion S5™ XL sequencers. The chipselectronically detect polymerase-drivenbase incorporation without the use offluorescence. By eliminating the use of anoptical detection system, this advance innext-generation sequencing technologyallows for rapid sequencing times of aslittle as 2.5 hours for 200 bp sequencing,with little hands-on time from sample tosequencing data.

Helpful tools

Tool 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 B Supplemental informationHelpful tools B


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/4488146?ICID=search-product




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

https://genome.ucsc.edu/

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 Repeat Maskerannotation tracks. Although this allows you to determine if the regions were filteredbased on either repeat regions or SNPs under the 3' primer, the designer also filters forother reasons. This section of the User Guide offers some suggestions for using otherbioinformatics tools to determine the quality of the design, and for diagnostic andresearch purposes.

One common reason AmpliSeq™ designer may have filtered the region would be thatit is a non-unique sequence and would hybridize in other areas of the genome andthus be deleterious to your reaction. BLAST, the Basic Local Alignment Search Tool,finds regions of local similarity between sequences.

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

Minimize off-targethybridization

Appendix B Supplemental informationAdvanced features and toolsB


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

3. Select Human, running a nucleotide query.

Appendix B Supplemental informationAdvanced features and tools B


If the search returns multiple results for that query, the designer has filtered thisto minimize off-target hybridization.

The designer filters regions that have either very high or very low GC % content.

1. Using the UCSC Table Browser, select the Mapping and Sequencing Tracks andGC content track options.

Determine GCcontent

Appendix B Supplemental informationAdvanced features and toolsB


2. You can enter to search for a specific region, or chose to upload your designedbed file to the Browser.The output will be in a 5-basepair sliding windows where you can determineregions that are below 20% or above 80% in GC content.

You can use the coverageAnalysis Plugin on your Torrent Browser to determinecoverage uniformity of your sequence run. Details on using the coverageAnalysisPlugin can be found on the Ion Community.

AddcoverageAnalysisPlugin

Appendix B Supplemental informationAdvanced features and tools B


Documentation and support

Related documentation

Document Description

Ion AmpliSeq™ Library Kit Plus User Guide(Pub No. MAN0006735)

This user guide contains completeprotocols for preparing libraries from DNAand RNA,and covers the following products:

• Ion AmpliSeq™ Library Kits 2.0 (Cat.No. 4475345, 4480441, 4480442)

• Ion Library Equalizer™ Kit (Cat. No.4482298)

• Ion AmpliSeq™ Ready-to-Use Panels(various Cat. Nos.)

• Ion AmpliSeq™ Made-to-Order andCommunity Panels

Ion AmpliSeq™ Library Kit Plus User Guide(Pub. No. MAN0017003)

This user guide contains completeprotocols for preparing libraries from DNAand RNA, and covers the followingproducts:

• Ion AmpliSeq™ Library Kit Plus (Cat.No. 4488990)

• Ion Library Equalizer™ Kit (Cat. No.4482298)

• Ion AmpliSeq™ Ready-to-Use Panels(various Cat. Nos.)

• Ion AmpliSeq™ Made-to-Order andCommunity Panels

• Ion AmpliSeq™ On‑Demand Panels

Qubit™ dsDNA HS Assay Kits User Guide(Pub. No. MAN0002326)

These user bulletins describe sensitivemethods for quantifying gDNA samples forlibrary preparation.

Demonstrated Protocol: SampleQuantification for Ion AmpliSeq™ LibraryPreparation Using the TaqMan® RNAse PDetection Reagents Kit (Pub. No.MAN0007732)

Ion Library TaqMan® Quantitation Kit UserGuide (Pub. No. MAN0015802)

This user guide covers a recommendedmethod for quantifying Ion AmpliSeq™

library concentration.


Customer and technical support

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

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

• Order and web support• Product documentation, including:

– User guides, manuals, and protocols– Certificates of Analysis– Safety Data Sheets (SDSs; also known as MSDSs)

Note: For SDSs for reagents and chemicals from other manufacturers,contact the manufacturer.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forthin the Life Technologies' General Terms and Conditions of Sale found on LifeTechnologies' website at www.thermofisher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact LifeTechnologies at www.thermofisher.com/support.

Documentation and supportCustomer and technical support


http://thermofisher.com/support

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

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

http://www.thermofisher.com/support

thermofisher.com/support | thermofisher.com/askaquestion

thermofisher.com

24 May 2017

http://thermofisher.com/support

mailto:thermofisher.com/askaquestion

http://thermofisher.com

ion ampliseq designer: getting started - amazon s3 · 2017-06-02 · in ion ampliseq™ designer,...

Documents