Ion AmpliSeq Designer: Getting Started

Transcription

1 Ion AmpliSeq Designer: Getting Started USER GUIDE Publication Number MAN Revision E.0 For Research Use Only. Not for use in diagnostic procedures.

2 Manufacturer: Life Technologies Corporation Carlsbad, CA USA Toll Free in USA The information in this guide is subject to change without notice. DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Revision history: MAN Revision Date Description 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 accept the terms and conditions of all applicable Limited Use Label Licenses. Trademarks: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered trademark of Roche Molecular Systems, Inc., used under permission and license Thermo Fisher Scientific Inc. All rights reserved.

3 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 Copy existing amplicons Known limitations of Copy amplicons Basic and biological filtering Filtering of repeat regions Biological filtering GC content Repeat regions and RepeatMasker filtering CHAPTER 2 Start an Ion AmpliSeq On Demand Panel design Start a new On-Demand design CHAPTER 3 Start an Ion AmpliSeq Made-to-Order Panel design Start a new DNA or RNA panel design Start an RNA gene fusion design Add gene expression assays Ion AmpliSeq Designer: Getting Started User Guide 3

4 Contents APPENDIX A Troubleshooting APPENDIX B Supplemental information Padding coordinates or create a BED file from list of variants Create a BED file for input into Ion AmpliSeq Designer Work from a list of COSMIC IDs Start from a list of dbsnp target identifiers Use the UCSC Genome Browser to create a BED file with Padded Exons Reference FASTA sequence Known polymorphism BED file Pipeline details The design pipeline Tiling algorithm Pooling algorithm Advantages of pooling and tiling Hotspots pipeline Frequently asked questions (FAQs) General Ion AmpliSeq Designer FAQs Ion AmpliSeq On Demand Panels FAQs Related Ion sequencing products Helpful tools Advanced features and tools Minimize off-target hybridization Determine GC content Add coverageanalysis Plugin Documentation and support Related documentation Customer and technical support Limited product warranty Ion AmpliSeq Designer: Getting Started User Guide

5 1 Getting started with Ion AmpliSeq Designer Welcome to Ion AmpliSeq Designer! This chapter will describe some procedures and tools used in Ion AmpliSeq Designer, and will show how to browse predesigned Ready-to-Use and Community Panels. Chapters 2 and 3 will cover how to design and order 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. The home page shows the options available for designing and ordering Ion AmpliSeq panels, and shows the latest product news. Three design and ordering options are now available: Ready-to-Use Panels: predesigned DNA and RNA panels for germline or somatic analysis, 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 reaction packs. Made-to-Order Panels: DNA and RNA designs for germline or somatic analysis of any genome, ordered in large reaction packs. Made-to-Order Panels were formerly known as Custom Panels. Ion AmpliSeq Designer: Getting Started User Guide 5

6 1 Chapter 1 Getting started with Ion AmpliSeq Designer Browse Ion AmpliSeq Ready-to-Use and Community Panels Browse Ion AmpliSeq Ready-to-Use and Community Panels 1. Sign in to Ion AmpliSeq Designer with your Thermo Fisher Scientific log in information. 2. To browse pre-designed Ion AmpliSeq panels, click DNA or RNA in the Readyto-Use Panels section. A table displaying research panels and their descriptions appears. 6 Ion AmpliSeq Designer: Getting Started User Guide

7 Chapter 1 Getting started with Ion AmpliSeq Designer Browse Ion AmpliSeq Ready-to-Use and Community Panels 1 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 details by clicking the More and Review Panel buttons. Click Download panel files to obtain panel files required for analysis of your sequencing results. Ion AmpliSeq Designer: Getting Started User Guide 7

8 1 Chapter 1 Getting started with Ion AmpliSeq Designer Navigation bar You can also browse pre-designed Community Panels in the same research areas by clicking the Pre-designed by our Community button in the Made-to-Order Panels section on the right side of the home screen. Navigation bar The navigation bar provides easy access to areas of interest 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. 8 Ion AmpliSeq Designer: Getting Started User Guide

9 Chapter 1 Getting started with Ion AmpliSeq Designer Chip Calculator 1 Chip Calculator The Chip Calculator provides recommendations for maximum sample library loading per chip for a given panel, chip, and coverage (500X for somatic samples, and 30X for germline samples). You can use the calculator as a guide for library loading for Readyto-Use panels, and to aid in the design of a Made-to-Order panel. Access the Chip Calculator by clicking the Review panel button of a Ready-to-Use or Community panel, 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 applicationspecific. Also note that instrument selection changes the Chip options in the lower table. Ion AmpliSeq Designer: Getting Started User Guide 9

10 1 Chapter 1 Getting started with Ion AmpliSeq Designer Chip Calculator The panel-specific chip calculator contains pre-populated fields pertaining to that panel. 10 Ion AmpliSeq Designer: Getting Started User Guide

11 Chapter 1 Getting started with Ion AmpliSeq Designer Create and manage reference genomes 1 Create and manage reference genomes In Ion AmpliSeq Designer, you can use various human, animal, and plant reference genomes to build your panels. You can also upload your own. The following steps describe how to upload a custom reference. 1. From the navigation bar, select Preloaded Genomes. The Design References screen appears and Public Genomes are displayed. If you have previously uploaded custom references, click the Custom References tab to view them. Note: You cannot select a public genome at this point. Public genomes are the available choices when you start a new design. Here, the list of public genomes is for informational purposes only. 2. To upload a new custom reference, click the Add reference button. Ion AmpliSeq Designer: Getting Started User Guide 11

12 1 Chapter 1 Getting started with Ion AmpliSeq Designer Create and manage reference genomes 3. Fill in the required information on the Add a custom reference screen, then click Select file under Reference sequences (FASTA file). Navigate to the location of the FASTA file on your drive, then select it Reference Name Must be composed of US-ASCII letters, numbers, and spaces, between 3 and 32 characters in length. 12 Ion AmpliSeq Designer: Getting Started User Guide

13 Chapter 1 Getting started with Ion AmpliSeq Designer Create and manage reference genomes 1 2 Associated organism for primer specificity check Click to view the dropdown menu containing list of organisms. If your data are associated with one of our supported organisms, providing this information can improve primer specificity to your custom reference by favoring primers with few optimal binding sites in the consensus sequence. Primer specificity check refers to the process of identifying potential primer mispriming events. Primers with high number of potential mispriming events are avoided in our designs. 3 Reference source (Recommended) Name of the database/source of the DNA sequence. 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 paste the 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 custom reference FASTA file with high polymorphism (that is, SNPs, indels, or other variation). Ion AmpliSeq Designer minimizes primer overlap with these regions. This file is optional. See the Appendix for specifications on creating and formatting BED files for uploading. 4. If appropriate, click Select file under Known polymorphism (BED) file to upload the polymorphism BED file. 5. After the upload progress bars complete, click Save. When the upload procedure finishes, select the Custom References tab to view your uploaded reference. Ion AmpliSeq Designer: Getting Started User Guide 13

14 1 Chapter 1 Getting started with Ion AmpliSeq Designer Create and manage reference genomes 6. Click the custom reference name to show more information. 14 Ion AmpliSeq Designer: Getting Started User Guide

15 Chapter 1 Getting started with Ion AmpliSeq Designer Create and manage reference genomes 1 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 are permanently associated with this assigned custom reference genome. To make changes, 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 are still downloadable. Ion AmpliSeq Designer: Getting Started User Guide 15

16 1 Chapter 1 Getting started with Ion AmpliSeq Designer Copy existing amplicons 8. When building your custom panel, click Custom Reference, then select your custom reference from the dropdown list. Copy existing amplicons Amplicons from one or more Ion AmpliSeq Ready-to-Use Panels, Community Panels, and/or previous Made-to-Order designs can be copied and resubmitted into the pipeline to generate new designs, without needing to calculate the position of the amplicon. 1. Create a new draft design from the home page, then determine the source of the amplicons you want to copy from (Ready-to-Use Panels, Community Panels, and/or previous designs). To copy amplicons from Ready-to-Use and/or Community Panels: a. Click the Browse by DNA or RNA button, or the Pre-designed by our Community button, then select your panel type. b. Select the panel that you are interested in subsetting from, then click Review panel. 16 Ion AmpliSeq Designer: Getting Started User Guide

17 Chapter 1 Getting started with Ion AmpliSeq Designer Copy existing amplicons 1 2. Check the box to select your gene of interest, then click Copy amplicons to copy all 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 the dropdown, or enter a new design name. Note: If you are copying amplicons into a pre-existing design, amplicons must 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 gene row 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 new design: Note: Follow these same steps to manually copy and add specific Amplicon IDs from previous designs and/or additional panels. You can also manually enter predesigned genes(s)/region(s) or upload a list of amplicons/gene using 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 and amplicons from custom designs mixed and matched with amplicons from other custom designs or Ready-to-Use and Community Panels. Ion AmpliSeq Designer: Getting Started User Guide 17

18 1 Chapter 1 Getting started with Ion AmpliSeq Designer Basic and biological filtering Known limitations of Copy amplicons Custom amplicons from a private design that is shared with you by a colleague cannot be copied using the "Add Amplicon By ID" form or the "Upload File" method. Amplicons can only be copied using the Copy amplicons button and dialog in the UI. The following DNA panels are not compatible for use with the Copy amplicons function: A HID-Ion AmpliSeq Identity Panel A 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 in our database. For RNA designs, use the Copy Targets function to create Made-to- Order RNA designs containing the same amplicons. Basic and biological filtering Filtering of repeat regions The human genome is heavily populated with repeat regions that make designing primers difficult, a well known challenge in PCR design. The Ion AmpliSeq Designer pipeline has been developed to deliver the most robust set of amplicons it can generate. The pipeline specifically excludes amplicons that are placed in repeat elements or other hypervariable regions to generate the best possible outcome for actual amplicon coverage when used in a reaction. A focus in our Research & Development department is to better understand the properties of repeat regions to allow primer placement in these regions to achieve higher 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 a program that screens DNA sequences for interspersed repeats and low complexity DNA sequences, and is an annotation track that is available through the UCSC Genome 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 visual representation 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 data appears 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) 18 Ion AmpliSeq Designer: Getting Started User Guide

19 Chapter 1 Getting started with Ion AmpliSeq Designer Basic and biological filtering 1 Biological filtering GC content Repeat regions and RepeatMasker filtering While G-C bonds contribute more to the stability, and therefore increased melting temperature, of primer/template binding than do A T bonds, it is important to note that two primer / template complexes with similar or even identical GC content can result in a different melting temperature because of base order influence on overall stability. GC-rich regions for the target DNA are difficult to amplify, and are generally avoided when defining an in silico algorithm. When submitted as a manual target region with coordinates: chr13: , the resulting coverage was 38.31% with 4 pools and 611 amplicons. In closer examination of the design results, it is apparent that the majority of this region is interspersed with repeat elements. An attempt to re-design the region by input type Gene + UTR results in coverage of 92.58% with 4 pools and 60 amplicons. Ion AmpliSeq Designer: Getting Started User Guide 19

20 2 Start an Ion AmpliSeq On Demand Panel design Ion AmpliSeq On-Demand Panels represent a new way to design panels for use in inherited disease (germline mutation) research applications. Ion AmpliSeq On-Demand Panels can be designed by selecting genes using a content selection engine, or uploading your own gene list. Each panel consists of two DNA primer pools, 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 9,000 amplicons. The gene 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-in Panel. This chapter covers how to design an Ion AmpliSeq On-Demand Panel using the features 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 login information. If you are already signed in but not on the home page, click the Home icon in the navigation bar to return to the home page, then click the Disease Research Areas button. 20 Ion AmpliSeq Designer: Getting Started User Guide

21 Chapter 2 Start an Ion AmpliSeq On Demand Panel design Start a new On-Demand design 2 2. On the Browse Disease Research Areas page, scroll to an area of interest, then click > 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 the disease category. Ion AmpliSeq Designer: Getting Started User Guide 21

22 2 Chapter 2 Start an Ion AmpliSeq On Demand Panel design Start a new On-Demand design 3. When you have found a Disease Research Area that most closely matches your interest, 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 total number of genes or amplicons in your selections exceeds these limits, you need 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 in the 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 22 Ion AmpliSeq Designer: Getting Started User Guide

23 Chapter 2 Start an Ion AmpliSeq On Demand Panel design Start a new On-Demand design 2 order the panel copy or clone an ordered and locked panel to make further changes 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 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 by score, a ranking of the relationship between the disease and gene, or alphabetically 9 Genes available as On-Demand or not available as On-Demand (Spike-in Panel) Ion AmpliSeq Designer: Getting Started User Guide 23

24 2 Chapter 2 Start an Ion AmpliSeq On Demand Panel design Start a new On-Demand design 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 to generate a Spike-in Panel. 24 Ion AmpliSeq Designer: Getting Started User Guide

25 Chapter 2 Start an Ion AmpliSeq On Demand Panel design Start a new On-Demand design 2 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. Ion AmpliSeq Designer: Getting Started User Guide 25

26 3 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 new design is a simple process. Sign in to Ion AmpliSeq Designer with your Thermo Fisher Scientific login information. If you are already signed in but not on the home page, click the Home icon in the navigation bar to return to the home page, then click Genes, Regions, or Amplicons. 26 Ion AmpliSeq Designer: Getting Started User Guide

27 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start a new DNA or RNA panel design 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), DNA Hotspot designs (single-pool), RNA Gene Expression designs (single-pool), or RNA Gene Fusion designs (multi-pool). Clicking the application type filters the compatible genomes shown. Note: Custom reference genomes are currently only compatible with DNA designs. RNA Gene designs are only compatible with the human genome. 3. Select a genome to use as reference. For custom references, click Custom Reference. 4. Click Next: Add Targets to proceed. Ion AmpliSeq Designer: Getting Started User Guide 27

28 3 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start a new DNA or RNA panel design 5. Three options for adding targets are available: Add Gene/Region, Add Amplicons by ID, and Upload File. Option Add Gene/Region Allows various manual options: Description 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 box appears after each to let you know if the target was added successfully. 4. When finished, click Submit targets. Add Amplicon by ID Upload File Allows you to enter amplicon IDs assigned to specific genomic coordinates. Allows you to upload genomic coordinates of several targets at a time, 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 the table, or remove them by checking their checkbox(es), then clicking the Delete button. 7. (Optional) Click Export targets to download your targets into a CSV file. 28 Ion AmpliSeq Designer: Getting Started User Guide

29 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start a new DNA or RNA panel design 3 8. To submit your panel, click Submit targets. Two designs can be submitted at a time. 9. Confirm your submission. When confirmed, you see a confirmation message and receive an confirming the design submission. 10. When the Assay Design results are ready, you receive an instructing you to review the results in Ion AmpliSeq Designer. Click the View results link provided in the to be directed to the results page (or navigate to AmpliSeq.com, then click the notification or navigate to the completed design using the My Designs tab). Ion AmpliSeq Designer: Getting Started User Guide 29

30 3 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start a new DNA or RNA panel design 11. Use the following fields to review your order: 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 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 to another Ion AmpliSeq Designer account holder. Note: Sharing your design also makes your custom reference available for review and downloading by anyone to whom you 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. 30 Ion AmpliSeq Designer: Getting Started User Guide

31 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start an RNA gene fusion design 3 Start an RNA gene fusion design To make RNA Gene Fusion panels with a combination of Gene Fusion and Gene Expression 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 with targets of your choice. For panels requiring two pools, the GEX assays are split between the two pools. 1. If you are already on AmpliSeq.com working in another application, click the Home icon in the navigation bar to return to the main page. 2. Click the Genes, Regions or Amplicons button in the Made-to-Order Panels section, then enter a Design Name and, optionally, details. Ion AmpliSeq Designer: Getting Started User Guide 31

32 3 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start an RNA gene fusion design 3. Select RNA Gene Fusion designs (multi-pool) button, in application type. Note: Human (RefSeq/Ensembl) is the only genome that is permitted with this panel type. 32 Ion AmpliSeq Designer: Getting Started User Guide

33 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start an RNA gene fusion design 3 4. Click Next: Add Targets. At the top of the Review Draft Designs screen, you can Add Fusions by Symbol Pair or upload them from a file. Click "Input Specifications" link for details. At the bottom of the screen, you see two tabs, Fusions to add gene fusion targets, and Genes to add gene expression targets. These tabs represent the two stages that are mentioned in the introduction 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. Ion AmpliSeq Designer: Getting Started User Guide 33

34 3 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start an RNA gene fusion design b. Select the desired gene pair. c. Add the fusions by clicking the checkbox to the left of each target, then click Add 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 saved successfully" in green text, or "# duplicate fusions ignored" in red text. 34 Ion AmpliSeq Designer: Getting Started User Guide

35 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start an RNA gene fusion design 3 Add gene expression assays Now add your gene expression assays. Every pool is required to have 12 gene expression assays, and we have pre-selected them to get you started. To pick your own gene expression assays, delete the proposed ones and add your own gene expression 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 HGNCapproved symbol) as in the following example, or valid RNA RefSeq accession numbers, then click Add target. Alternatively, you can upload your own gene expression assays. Click the Input Specifications link in the software user interface for more details and a CSV template 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 gene expression 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 also sent to you via When your fusion results are ready, you receive an instructing you to review the results in Ion AmpliSeq Designer. Note: You may need to check your spam folder and move the to your Inbox to enable its links. Ion AmpliSeq Designer: Getting Started User Guide 35

36 3 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start an RNA gene fusion design 7. Click the link provided in the , or go to the Ion AmpliSeq Designer website and navigate to My Designs4RNA. 8. Click the name of your design in the Design column. 36 Ion AmpliSeq Designer: Getting Started User Guide

37 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start an RNA gene fusion design 3 9. Review results on the Fusions tab, then make changes if needed by clicking Copy Targets and submitting a new design with your changes 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. Ion AmpliSeq Designer: Getting Started User Guide 37

38 3 Chapter 3 Start an Ion AmpliSeq Made-to-Order Panel design Start an RNA gene fusion design 11. Click the Gene link to view compatible transcripts. 38 Ion AmpliSeq Designer: Getting Started User Guide

39 A Troubleshooting For complete Ion AmpliSeq library troubleshooting, see the Ion AmpliSeq Library Kit 2.0 User Guide (Pub. No. MAN ), or the Ion AmpliSeq Library Kit Plus User Guide (Pub. No. MAN ), available at thermofisher.com. The following table can help determine probable cause and recommended action for several performance observations. Observation Possible cause Recommended action Short amplicons are under-represented Purification was poor. Vortex AMPure XP Reagent thoroughly before use, and be sure to dispense the full volume. Long amplicons are under-represented (short library reads) Sample DNA or RNA was degraded. PCR was inefficient. Too few nucleotide flows were used. Sample was over-treated with FuPa Reagent. Denaturation temperature was too high. 100% ethanol is difficult to pipet accurately; it is essential to pre-wet pipette tips. In post-ligation library purification, increase AMPure XP Reagent volume from 45 μl (1.5X) to 50 μl (1.7X). Use an FFPE assay design for degraded or low quality samples. Double the anneal and extend time. Use an appropriate number of flows to sequence through amplicons. Add no more than 2 µl FuPa Reagent per 20 µl target amplification reaction. Keep the plate on ice during FuPa Reagent addition, then transfer to a preheated thermal cycler immediately. Use a 97 C enzyme activation/denaturation temperature instead of 99 C in target amplification reactions. Ion AmpliSeq Designer: Getting Started User Guide 39

40 A Appendix A Troubleshooting Start an RNA gene fusion design Observation Possible cause Recommended action AT-rich amplicons are under-represented Example of loss of AT-rich amplicons. Within the Coverage Analysis Plugin, amplicon representation is plotted by GC content for an Ion AmpliSeq Panel. Amplicons with 23-50% GC show an excess failure rate (less than 20% of the mean read depth). GC-rich amplicons are under-represented Example of loss of GC-rich amplicons. Within the Coverage Analysis Plugin, amplicon representation is plotted by GC content for an Ion AmpliSeq Panel. Amplicons with 60-80% GC show an excess failure rate (less than 20% of the mean read depth). Target amplification was inefficient. Digested amplicons were denatured. Digestion was inefficient. Denaturation was inadequate during target amplification. Target amplification was inefficient. Library amplification was inefficient. Sample was over-treated with FuPa Reagent. Double the anneal/extend time in the target amplification reaction. Decrease the anneal/extend temperature of the target amplification reaction from 60 C to 58 C. Do not exceed 60 C during the amplicon digestion step. Increase amplicon digestion times to 20 minutes for each step. Use a calibrated thermal cycler. Increase the anneal/extend temperature of the target amplification reaction from 60 C to 62 C for the first two cycles of the target amplification reaction Do not amplify the library (not required for qpcr quantification). Add no more than 2 µl FuPa Reagent per 20 µl target amplification reaction. Keep the plate on ice during FuPa Reagent addition, then transfer to thermal cycler immediately. 40 Ion AmpliSeq Designer: Getting Started User Guide

41 B Supplemental information Padding coordinates or create a BED file from list of variants Reference FASTA sequence Known polymorphism BED file Pipeline details Hotspots pipeline Frequently asked questions (FAQs) Related Ion sequencing products Helpful tools Advanced features and tools Padding coordinates or create a BED file from list of variants Create a BED file for input into Ion AmpliSeq Designer 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 or dbsnp identifiers, use the UCSC Table Browser of the UCSC Genome Browser. Make sure to select genome and assembly as Human, (GRCh37/hg19). Work from a list of COSMIC IDs 1. Select All Tracks in the group drop-down list, and COSMIC for the track. 2. Choose the COSMIC option in the table list. Ion AmpliSeq Designer: Getting Started User Guide 41

42 B Appendix B Supplemental information Padding coordinates or create a BED file from list of variants Start from a list of dbsnp target identifiers 1. Select the Variation and Repeats group in the drop-down menu. The track and table sections are then populated with available SNP databases from dbsnp. 2. Choose the genome radio button for the region selection. Click on either the paste list or upload list button next to identifiers. 3. Either upload a text file listing your COSMIC or dbsnp ids or simply paste in the list and click submit. For the output format, select BED - browser extensible data. 42 Ion AmpliSeq Designer: Getting Started User Guide

43 Appendix B Supplemental information Padding coordinates or create a BED file from list of variants B 4. After clicking on the get output button, the Output as BED screen allows for padding 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. Ion AmpliSeq Designer: Getting Started User Guide 43

44 B Appendix B Supplemental information Padding coordinates or create a BED file from list of variants Use the UCSC Genome Browser to create a BED file with Padded Exons 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 be directed to the Output refgene as BED page. 2. Select options to add upstream or downstream base padding, or bases at each end of the exons. 3. Additionally, select to create the BED file to include 5' or 3' UTR Exons or CDS. 44 Ion AmpliSeq Designer: Getting Started User Guide

45 Appendix B Supplemental information Reference FASTA sequence B 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) containing the sequence(s). The maximum file size allowed for upload is 1 GB after decompression. b. Copying and pasting the sequences in the text area available after clicking on the link "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 an identifying "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 by at most 64 ASCII characters. Allowed characters are A-Z, a-z, 0-9, "_" and "-", with NO SPACES between them. Since 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 accepted for representing a sequence are "A," "C," "G," "T," and "N" (lower case versions are also allowed for representing low complexity regions). Although a sequence can be just one or multiple lines of different size after the header, it is traditional to use separate lines of 50 or 60 characters in length. Sequence size: The minimum length of a sequence is 160 bp: allowing 60 bp for minimum insert size, plus 50 bp upstream and 50 bp downstream to serve as a design buffer for primer positioning during amplicon design; however, the recommended upstream and downstream context buffer sequence for optimal designs is 1,000 bp. Known polymorphism BED file The known polymorphism BED file indicates regions of the sequences in the custom reference FASTA file with high polymorphism (i.e., SNPs, indels, or other variation). Ion AmpliSeq Designer will minimize primer overlap with these regions. This file is optional. 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 are chrom, 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 indicating the region to target in the sequence identified by the ID in the chrom field. chromstart and chromend are relative to the sequence of the FASTA record corresponding to the given ID. Ion AmpliSeq Designer: Getting Started User Guide 45

46 B Appendix B Supplemental information Pipeline details 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_ contig_ contig_ Pipeline details When you use Ion AmpliSeq Designer, the tiling and pooling algorithms run behind the scenes to transform your submitted design into an orderable Custom Ion AmpliSeq kit. The following sections describe the Ion AmpliSeq tiling and pooling processes. 46 Ion AmpliSeq Designer: Getting Started User Guide

47 Appendix B Supplemental information Pipeline details B The design pipeline The pipeline is optimized to find the highest coverage with the least number of amplicon pools. In an attempt to design all primer pairs exhaustively, the pipeline creates an exhaustive search by sliding a tiling window one-base at a time across the entire 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 allowed anywhere 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 Tiling algorithm The tiling algorithm selects a subset (a tiling) of the input amplicons that meets the following 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. Ion AmpliSeq Designer: Getting Started User Guide 47

48 B Appendix B Supplemental information Hotspots pipeline The output from the tiling algorithm is used as input for the pooling algorithm. Improvements in the tiling algorithm have significantly increased the in silico coverage of the designs. The users are enabled to choose between in silico and assay performance. Pooling algorithm The pooling algorithm assigns each tile (amplicon) to a pool, subject to requirements that 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, the algorithm puts the amplicon into the first compatible pool that maximizes the distance 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 and returns to step 3. c. If a new pool cannot be added, the algorithm puts the amplicon into the overflow (unpooled) list. Advantages of pooling and tiling The tiling algorithm ensures that the PCR pools cover as much of the targets as do 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 require two or more pools of primers. Designs targeting small genomic regions such as SNPs and small indels are well suited to fit in a single pool because those regions can be covered by non-overlapping amplicons. In standard designs the "tiling" stage of the pipeline is when amplicons are selected to cover 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 a set of overlapping amplicons that, if put together, are prone to interact with each other during the sequencing process. The "pooling" stage is when as many pools as needed are created to avoid these interactions. The hotspots pipeline includes special algorithms that allow in the pooling stage the creation of a single pool of primers with maximum coverage for targets as small as one base, and as large as 50 bases. 48 Ion AmpliSeq Designer: Getting Started User Guide

49 Appendix B Supplemental information Hotspots pipeline B The idea behind the hotspots designer can be better understood by looking at the following diagram in which the long line represents a genomic region; a, b, and c are the amplicons available for covering that region. The red portions of the amplicons represent the primers. c a b 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 primers at 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 to be in one pool would eliminate amplicon a or b resulting in reduced coverage for the one-pool solution. Using amplicons c and b is not an option because they fail to cover a portion of the region between the arrows. In contrast, for a hotspots panel, the targets are small genomic regions (marked with dots over the line representing the genome), the hotspots pipeline would actually select non-overlapping amplicons c and b to cover the submitted targets. Non-overlapping amplicons can be put together in a single pool of primers. Ion AmpliSeq Designer: Getting Started User Guide 49

50 B Appendix B Supplemental information Frequently asked questions (FAQs) Frequently asked questions (FAQs) General Ion AmpliSeq Designer FAQs 1. How does the software accommodate intronic regions? When you submit a gene to design, only exons are used as targets. To design across the whole gene (exons and introns), submit the start and end coordinates of 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 describing the genome coordinates. 3. What is the level of overlap among the primers? Are the overlapping primers in the same tube? Primers in the same tube do not overlap. As our product line evolves in the future, a small overlap might be possible. 4. For Ion AmpliSeq Designer, are primer sets designed automatically (with a computer program), without interrogation from a research scientist? The process is an automated pipeline, optimized to provide the maximum coverage with reliable primer sets. 5. How are the Ion AmpliSeq Custom designs verified? Each primer pool goes through a rigorous process to meet strict design specifications. During the design of our pipeline, we verified a substantial number 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 working with 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 bp range? No, you do not have to select the gene coordinates (BED file) to be bp. Ion AmpliSeq Designer designs the primer pairs, and provides the appropriate BED file and primer sequences to generate amplicons approximately 200-bp in length. 8. If I submit two continuous regions ( bp range each) combined as one BED file, is it possible to get the designed primers for the overlapping region? If overlapping regions are submitted to the design pipeline, internally the region is concatenated and treated as a single region for design, therefore there is no overlap. The two regions are reported back in the UI as submitted. Although it is possible that an amplicon is prorated twice, one time in each original region, this amplicon (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 the aligned sequence section, we have 0.00% coverage of the genome. Is this result because the fraction of the genome that is covered is so low compared to the hg19 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 so low 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 large amplicon. The pooler algorithm in the pipeline separates primers into separate pools to minimize this occurrence. 50 Ion AmpliSeq Designer: Getting Started User Guide

51 Appendix B Supplemental information Frequently asked questions (FAQs) B 11. The BED file specifications state that in a BED file the chrstart number is zeroindexed and the chrend number is not included in the feature. Are you following this convention for upload and are the numbers shown in the designer 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 position of the feature in the chromosome or scaffold. The chromend base is not included in the display of the feature. For example, the first 100 bases of a chromosome are defined as chromstart=0, chromend=100, and span the bases numbered Can you describe further how the Ultraplex technology works? Development work from over a decade allows us to produce primer designs that allow simultaneous amplification of many amplicon targets. A unique chemistry has been developed for Ion AmpliSeq libraries that allows removal of any primer-dimer that is formed, along with most of the primer itself from the amplified template. This removal makes sequencing efficient by not wasting bases on non-informative primer sequence, and allows for clean sequencing reactions. 13. Do your designs account for the presence of pseudogenes? Yes. The pipeline first attempts to design primers that only match the target, and not the pseudogene (or duplicate) versions. If the target gene is not covered in the first rounds of primer selection, then the match parameters are relaxed, for the sake of coverage, in later rounds, attempting to maintain the uniqueness of the inserts. 14. If two amplicons overlap, do the primers produce a large product in addition to two small products? The pooling step in the design is optimized to minimize the interference between overlapping amplicons. Overlapping amplicons are segregated into different pools. 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 and chr6_cox_hap Which sequence versions does Ion AmpliSeq Designer use in its computations? DNAHuman Genome* - Feb (hg19, GRCh37), Mouse Genome* - Dec (mm10, GRCm38), Gene targets correspond to RefSeq v63, Hotspots targets correspond to dbsnp v138 (for human and mouse) and COSMIC v68 (available only for human), RNAHuman RNA Canonical RefSeq Transcripts* - Feb (hg19, GRCh37), HGNC Database, HUGO Gene Nomenclature Committee (HGNC), EMBL Outstation - Hinxton, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, and Cambridgeshire, CB10 1SD, UK 11/2012 Ion AmpliSeq Designer: Getting Started User Guide 51

52 B Appendix B Supplemental information Frequently asked questions (FAQs) Ion AmpliSeq On Demand Panels FAQs 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 or 9,000 amplicons per panel due to manufacturing restrictions. As we continue to make improvements this limit is likely to increase and larger designs may be ordered. 2. Why is there a limit on the number of genes that I can add to my On-Demand panel? Because the order limit is set at 300 genes per panel, it becomes impractical to allow a large number of genes into the Grid or Table view, which need to be deselected to make the design orderable. For this reason we have introduced a limit 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 it has 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 locked after 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. This exports 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 Research Area (DRA)? Not directly. Currently we do not allow addition of DRA content to an existing design, only to new designs. The workaround is to create a new design with the desired DRA content, and then export the list of targets. You can then upload this list 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-Demand genes, and which you select are ordered. If you create a Spike-in Panel, that panel needs 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 the necessary files needed for analysis by Torrent Suite and Ion Reporter Software need to remain in sync with the material you ordered. If you need to edit your design, select the "Clone" option. A new IAD number is then assigned to your design, 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 gene symbols when creating an On-Demand Panel? The source of annotations is refgene, and the version that we are using is version v 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 of an On-Demand gene design. 11. Are UTR-only genes supported? What about pseudogenes? No, only genes containing CDS regions are supported. At this time, pseudogenes are not supported. 52 Ion AmpliSeq Designer: Getting Started User Guide

53 Appendix B Supplemental information Frequently asked questions (FAQs) B 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 design with collaborators. However, you can export the list of targets, and share that list with your collaborator. The design they create will be identical to yours if the list of targets is the same. 14. What is in silico coverage? In silico coverage is defined by the percentage of bases that are covered by the tiling of amplicons. This number is a computer-based calculation and should not be confused with experimental coverage, which represents the actual performance of the panel in the lab. 15. What is "Gene uniformity"? The number of reads spanning is counted for each base across all padded coding exons of a gene. An average value is calculated for all the bases, and the percentage of bases with read counts above 20% of the average value is defined as "Gene uniformity". 16. Have you checked for all possible gene combinations to test for primer-primer interactions? No, the number of possible combinations is astronomical and it is not possible to test for all possible combinations in the lab. What the team has done is use computer-based searches to reduce as much as possible the occurrence of primerprimer interactions. In addition, the genes have been synthesized in large gene batches, and we have observed less than 1% amplicon drop-out due to suspected primer-primer interactions. Disease Research Areas (DRAs) 1. What are the sources used for creating the associations for the various Disease Research Areas in the tool? The sources include DisGeNET ( Unified Medical Language System ( and Medical Subject Headings (MeSH). 2. What algorithm was used to create such associations? An in-house gene scoring algorithm was used to create these associations. Details of the algorithm are proprietary. 3. What does the "Score" mean? The "Score" ranks the relationship between a gene and a disease. It takes into account 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 create the 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 categories by clicking the box on the right, and then edit the gene content once the design is in 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. Ion AmpliSeq Designer: Getting Started User Guide 53

54 B Appendix B Supplemental information Frequently asked questions (FAQs) 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 one or 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 our algorithms that have aggregated the data. If your gene is not present, it is likely because the observed associations are below our threshold, or outside of the sources we used. Contact our support team (ampliseq-designs@thermofisher.com) if you are aware of strong evidence demonstrating 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 and Genome Sequencing. 10. What are "Newborn Screening Conditions" or "Newborn Screening" genes? These are genes associated with conditions listed in the Recommended Uniform Screening 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 observed for each amplicon of each targeted gene during our verification experiments. This track should only be used as general guidance of the likely performance observed when running the experiment. Values are likely to be different when a new 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 was not possible due to local environment complexity. We have made every effort to minimize 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 to 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 Grid View, click a gene and the IGV viewer is updated automatically and centered on that gene. 5. I ve noticed that occasionally, the "Expected coverage" track for an amplicon does not appear to contain information. Why is that? All amplicons in the design contain reads that are visualized in the "Expected coverage" track. If reads are not present, they are highlighted in the "Missed regions (if any)" track. It can happen that, if the number of reads covering an amplicon is relatively small in comparison to neighboring amplicons, the "Expected coverage" track appears empty. However, if you change the scale to a lower 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 in some instances primers can either bind less tightly, or bind off-target, thereby reducing the number of amplicon reads at the desired region. 54 Ion AmpliSeq Designer: Getting Started User Guide

55 Appendix B Supplemental information Frequently asked questions (FAQs) B Spike-in Panels 1. What are Spike-in Panels? Spike-in Panels are high concentration Made-to-Order Panels that are used to extend 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 Spikein Panel enables a user to sequence all the targets initially wanted in a single target 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 be included, and possible reduction in current expected coverage performance. The size of a compatible Spike-in Panel is limited to 123 amplicons per pool, for a total 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 novo for every order, and the number of reactions is typically large (between 750 to 3,000 reactions). On the other hand, On-Demand Panels have been optimized, pre-manufactured, tested and verified, and are available in small reaction number batches. On-Demand Panels also contain data that can be visualized in our integrated IGV viewer available in the Grid view. Ion AmpliSeq Designer: Getting Started User Guide 55

56 B Appendix B Supplemental information Related Ion sequencing products Related Ion sequencing products Link to product purchase page Ion AmpliSeq Library Kit 2.0 Ion AmpliSeq Library Kit Plus Ion AmpliSeq Sample ID Panel Ion Library Equalizer Kit Ion Xpress Barcode Adapters 1 16 Kit IonCode Barcode Adapters Kit Description Ion AmpliSeq DNA and RNA library preparation from targeted DNA panels for any genome. Ion AmpliSeq DNA and RNA library preparation from targeted DNA panels for any genome. Recommended for manual library preparation for Ion AmpliSeq On Demand Panels. The Ion AmpliSeq Sample ID Panel is a versatile, cost effective, and easy-to-use human SNP genotyping panel comprising 9 specially designed primer pairs that can be added to the multiplex PCR reaction to generate a unique ID during postsequencing analysis of research samples. The Ion Library Equalizer Kit is a simple and seamless bead-based solution replacing the need for library quantification and library dilutions for library normalization as required for any next generation sequencing workflow. The Ion Library Equalizer Kit helps reduce labor, reagent costs, and further simplifies the Ion semiconductor sequencing workflow for high sample-throughput or barcoded projects, as well as single or low sample number projects. The Ion Xpress Barcode Adapters 1 16 Kit provides a set of 16 unique barcode adaptors specifically designed and verified for optimal performance with the Ion PGM, Ion Proton, and Ion S5 /Ion S5 XL sequencers. When used in combination with the Ion AmpliSeq Library Kit 2.0 or the Ion AmpliSeq Library Kit Plus, this kit enables users to pool up to 16 libraries before template preparation, and then conduct multiplexed sequencing analysis, simplifying the Ion sequencing workflow for a wide range of applications. Use of this kit with other Ion Xpress Barcode Adapters kits allows pooling of up to 96 amplicon or fragment libraries. The IonCode Barcode Adapters Kit provides 384 different pre-mixed adapters in a convenient 96-well plate format. 56 Ion AmpliSeq Designer: Getting Started User Guide

57 Appendix B Supplemental information Helpful tools B Link to product purchase page Ion AmpliSeq Kit for Chef DL8 Ion PGM Hi Q View Chef Kit Ion PI Hi Q Chef Kit Ion 520 & Ion 530 Kit Chef Ion 540 Kit Chef Ion 318 Chip v2 BC Ion PI Chip Kit v3 Ion 530 Chip Kit Ion 540 Chip Kit Description The Ion AmpliSeq Kit for Chef DL8 enables automated Ion AmpliSeq DNA and RNA library preparation of up to 8 libraries per run on the Ion Chef System. Ion Chef template and sequencing kits enable robust and automated template preparation, chip loading, and sequencing of up to 400-base libraries on the Ion PGM, Ion Proton, Ion S5, and Ion S5 XL sequencers. Ion Chip kits contain chips for sequencing runs on the Ion PGM, Ion Proton, Ion S5, and Ion S5 XL sequencers. The chips electronically detect polymerase-driven base incorporation without the use of fluorescence. By eliminating the use of an optical detection system, this advance in next-generation sequencing technology allows for rapid sequencing times of as little as 2.5 hours for 200 bp sequencing, with little hands-on time from sample to sequencing data. Helpful tools Tool UCSC Table Browser UCSC Genome Browser Integrative Genomics Viewer (IGV) Description Use this program to retrieve the data associated with a track in text format, to calculate intersections between tracks, and to retrieve DNA sequence covered by a track. This site contains the reference sequence and working draft assemblies for a large collection of genomes. The Integrative Genomics Viewer (IGV) is a high-performance visualization tool for interactive exploration of large, integrated genomic datasets. It supports a wide variety of data types, including array-based and next-generation sequence data, and genomic annotations. Ion AmpliSeq Designer: Getting Started User Guide 57

58 B Appendix B Supplemental information Advanced features and tools Advanced features and tools Ion AmpliSeq Designer links the output designed BED file directly to the UCSC Genome Browser automatically configured with the dbsnp and Repeat Masker annotation tracks. Although this allows you to determine if the regions were filtered based on either repeat regions or SNPs under the 3' primer, the designer also filters for other reasons. This section of the User Guide offers some suggestions for using other bioinformatics tools to determine the quality of the design, and for diagnostic and research purposes. Minimize offtarget hybridization One common reason AmpliSeq designer may have filtered the region would be that it is a non-unique sequence and would hybridize in other areas of the genome and thus 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 the results of your design. 58 Ion AmpliSeq Designer: Getting Started User Guide

59 Appendix B Supplemental information Advanced features and tools B 2. Copy the sequence, and run a BLAST search on this sequence. 3. Select Human, running a nucleotide query. Ion AmpliSeq Designer: Getting Started User Guide 59

60 B Appendix B Supplemental information Advanced features and tools If the search returns multiple results for that query, the designer has filtered this to minimize off-target hybridization. Determine GC content 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 and GC content track options. 60 Ion AmpliSeq Designer: Getting Started User Guide

61 Appendix B Supplemental information Advanced features and tools B 2. You can enter to search for a specific region, or chose to upload your designed bed file to the Browser. The output will be in a 5-basepair sliding windows where you can determine regions that are below 20% or above 80% in GC content. Add coverageanalysis Plugin You can use the coverageanalysis Plugin on your Torrent Browser to determine coverage uniformity of your sequence run. Details on using the coverageanalysis Plugin can be found on the Ion Community. Ion AmpliSeq Designer: Getting Started User Guide 61