Ion AmpliSeq Designer: Getting Started

Transcription

1 Ion AmpliSeq Designer: Getting Started USER GUIDE Publication Number MAN Revision F.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, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Revision history: MAN Revision Date Description F.0 12 June 2018 Updated for the Ion AmpliSeq Designer v7.0 user interface Updated for Ion AmpliSeq HD panel design and ordering E.0 24 May 2017 Support added for Ion AmpliSeq On Demand Panels Updated for new Ion AmpliSeq Designer user interface D.0 5 October 2015 Updated screenshots for software user interface changes Added introduction sections for new user interface Support added for the Chip Calculator Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you 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 Get started with Ion AmpliSeq Designer... 5 Navigation bar... 7 Chip Calculator... 7 Create and manage reference genomes... 9 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 a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq On Demand Panel design Start a new Ion AmpliSeq Made-to-Order Panel design Start a new DNA Gene design Start a new DNA Hotspot design Start a new RNA Gene Expression design Start a new RNA Gene Fusion design Browse Ion AmpliSeq Ready-to-Use and Community Panels CHAPTER 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Gene design Start a new Ion AmpliSeq HD DNA Hotspot design Start a new Ion AmpliSeq HD RNA Gene Fusion design 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 custom BED file for input into Ion AmpliSeq Designer 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 Ion Chip capacities for Ion AmpliSeq DNA libraries sequenced at equal depths Ion Chip capacities for Ion AmpliSeq RNA libraries Frequently asked questions (FAQs) General Ion AmpliSeq Designer FAQs Ion AmpliSeq On Demand Panels FAQs Ion AmpliSeq HD 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 Get started with Ion AmpliSeq Designer Welcome to Ion AmpliSeq Designer! This chapter describes some procedures and tools used in Ion AmpliSeq Designer. Chapter 2 covers how to design and order Ion AmpliSeq On-Demand and Made-to-Order Panels, and browse and order predesigned Ready-to-Use Panels. Chapter 3 covers how to design and order ultra-high sensitivity Ion AmpliSeq HD panels. Ion AmpliSeq Designer: Getting Started User Guide 5

6 1 Chapter 1 Get started with Ion AmpliSeq Designer Trademarks Before you start a panel design or browse predesigned panels, sign in at AmpliSeq.com with your Thermo Fisher Scientific user name and password. The homepage shows the options available for designing and ordering panels, and has the latest product news. Four design and ordering options are available. On-Demand Ion AmpliSeq panels of optimized amplicons for germline analysis. Configurable to a specific human disease area, and ordered in small reaction packs. Made-to-Order Ion AmpliSeq Panels: DNA and RNA designs for germline or somatic analysis of any genome, ordered in large reaction packs. Note: Made-to-Order Panels were formerly known as Custom Panels. Ready-to-Use Ion AmpliSeq Panels: predesigned DNA and RNA panels for germline or somatic analysis, ordered in small reaction packs. Made-to-Order Ion AmpliSeq HD Panels: Ultra-high sensitivity DNA and RNA designs for germline or somatic analysis of the human genome, featuring dual barcoded amplicons and bidirectional sequencing. 6 Ion AmpliSeq Designer: Getting Started User Guide

7 Chapter 1 Get started with Ion AmpliSeq Designer Navigation bar 1 Navigation bar The navigation bar provides easy access to areas of interest Live Chat Chat with an Ion AmpliSeq Designer expert to get help and advice. 2 Home Takes you to the starting point for creating a panel design. 3 My Designs Browse the designs you have already created. 4 Chip Calculator Calculate the number of sample libraries you can load per chip given your panel, sample source, and chip type. 5 Notifications Provides updates of new features and messages from ampliseq.com. 6 Help Takes you to the customer help page. 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. Besides the navigation bar, you can also access the Chip Calculator by clicking the Review panel button of a Ready-to-Use panel, and in the Results Ready screen 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 7

8 1 Chapter 1 Get started with Ion AmpliSeq Designer Chip Calculator The panel-specific chip calculator contains pre-populated fields pertaining to that panel. Note: See Ion Chip capacities for Ion AmpliSeq DNA libraries sequenced at equal depths on page 71 and Ion Chip capacities for Ion AmpliSeq RNA libraries on page 72 for further information on chip capacity. 8 Ion AmpliSeq Designer: Getting Started User Guide

9 Chapter 1 Get 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 reference. 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 9

10 1 Chapter 1 Get 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. 10 Ion AmpliSeq Designer: Getting Started User Guide

11 Chapter 1 Get 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 Known polymorphism BED file on page 66 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. 6. When the upload procedure finishes, select the Custom References tab to view your uploaded reference. Ion AmpliSeq Designer: Getting Started User Guide 11

12 1 Chapter 1 Get started with Ion AmpliSeq Designer Create and manage reference genomes 7. Click the custom reference name to show more information. 12 Ion AmpliSeq Designer: Getting Started User Guide

13 Chapter 1 Get started with Ion AmpliSeq Designer Create and manage reference genomes 1 8. Click Edit 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. 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 13

14 1 Chapter 1 Get started with Ion AmpliSeq Designer Copy existing amplicons 9. 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 screen, 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. In the Ready-to-Use Panels tab, click the Browse by DNA or RNA button, or click the Pre-designed by our Community button in the Made-to-Order Panels tab. b. Select the panel that you are interested in subsetting from, then click Review panel. 14 Ion AmpliSeq Designer: Getting Started User Guide

15 Chapter 1 Get started with Ion AmpliSeq Designer Copy existing amplicons 1 2. In the row of your gene of interest, check the box to select the gene, then click Copy Amplicons to copy the selected amplicons to your new design. Note: Click anywhere in the row of a gene of interest to view a list of the amplicons included in that gene design. Ion AmpliSeq Designer: Getting Started User Guide 15

16 1 Chapter 1 Get started with Ion AmpliSeq Designer Copy existing amplicons 3. In the Copy Amplicons dialog, select either Copy checked target rows, or Copy unchecked targets instead of checked target rows, then click Copy amplicons to another design. Note: You can also click Download amplicon list to download a CSV file of the complete amplicon list to edit and upload to a custom design. You can copy the entire amplicon list from a panel by clicking the Copy Amplicons button for the panel in the panel list page, when the button is active. 4. In the Select Destination dialog, select a pre-existing design from the Destination dropdown list as a destination for the copied amplicons, or select New design..., then enter the name in the New design name field. 16 Ion AmpliSeq Designer: Getting Started User Guide

17 Chapter 1 Get started with Ion AmpliSeq Designer Copy existing amplicons 1 Click Copy amplicons to copy the amplicons to the selected design. Note: If you are copying amplicons into a pre-existing design, amplicons must be of the same genome, or in draft form only. 5. After you compile a list of targets for which you want to create a new design, click Submit targets, then confirm your submission. See Start a new DNA Gene design on page 26 for further information on how to submit Made-to-Order designs. Note: Subset designs can be combinations of predesigned regions and amplicons from custom designs mixed and matched with amplicons from other custom designs or Ready-to-Use and Community Panels. 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 dialogs in the UI. The following DNA panels are not compatible for use with the Copy Amplicons function: HID - Precision ID Identity Panel (Cat. No. A25643) HID - Precision ID Ancestry Panel (Cat. No. A25642) Ion AmpliSeq Exome RDY Panel 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. Amplicons that are greater than 200 bp in size cannot be copied to Ion AmpliSeq HD designs. Ion AmpliSeq Designer: Getting Started User Guide 17

18 1 Chapter 1 Get started with Ion AmpliSeq Designer Basic and biological filtering 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. Ion AmpliSeq 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) 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. 18 Ion AmpliSeq Designer: Getting Started User Guide

19 Chapter 1 Get started with Ion AmpliSeq Designer Basic and biological filtering 1 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 a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq On Demand Panel design Ion AmpliSeq On-Demand Panels represent an alternative 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 500 genes and/or 15,000 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 section covers how to design an Ion AmpliSeq On-Demand Panel using the features and tools available in Ion AmpliSeq Designer. 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 a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq On Demand Panel 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 a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq On Demand Panel 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 500, and the total number of amplicons cannot exceed 15,000. 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 each gene. create a Spike-in Panel by selecting Disease Research Area genes. 22 Ion AmpliSeq Designer: Getting Started User Guide

23 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq On Demand Panel 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 created/ordered 4 Export Targets generate an Excel.csv file of the target list 5 Upload File upload a.csv file containing a list of genes to include in a panel 6 Add Gene enter gene symbol to upload genes to panel design individually 7 Grid/Table toggle between the grid (shown) and table views of target genes 8 Sort By sort target list 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 a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq On Demand Panel design In the Grid view, click on 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 a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq On Demand Panel 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 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design Start a new Ion AmpliSeq Made-to-Order Panel design Start a new DNA Gene design If you are using one of the Ion AmpliSeq standard genome references, select the DNA Gene design option to design a panel to discover new variants as well as detect known SNPs and indels in your genes of interest. 1. Sign in to Ion AmpliSeq Designer with your Thermo Fisher Scientific login information. On the home page, click Made-to-Order Panels4Genes, Regions or Amplicons. 26 Ion AmpliSeq Designer: Getting Started User Guide

27 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2 2. In the Start a new AmpliSeq design screen, in the Name and details step, enter a Design Name and optional Details in the appropriate fields. 3. Select your Application type: DNA Gene designs (multi-pool). Clicking the application type filters the compatible genomes that are shown. 4. Select a genome to use as reference. For custom references, click Custom Reference. Note: Custom genome references are currently only compatible with DNA designs. 5. Click Next: Add Targets. Ion AmpliSeq Designer: Getting Started User Guide 27

28 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 6. 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, then select the gene from the dropdown list. 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). Note: Click Input Specifications for more information. Ion AmpliSeq Designer uploads the targets, checks them, and verifies regions. 7. For any highlighted erroneous target(s), either correct the coordinates inside the table, or remove them by checking their checkbox(es), then clicking Delete. 28 Ion AmpliSeq Designer: Getting Started User Guide

29 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2 8. Select an Exon padding value if different from the standard setting. 9. Select a DNA Type for the type of sample you will construct the library from: Standard DNA, FFPE DNA, or cfdna. Note: This setting affects amplicon length in the panel design. 10. (Optional) Click Export targets to download your targets into a CSV file. 11. To submit your panel, click Submit targets. Note: Two designs can be submitted at a time. 12. Click OK to confirm your submission. When confirmed, you see a confirmation message and receive an confirming the design submission. 13. 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 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 14. 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/pool), 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 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2 Start a new DNA Hotspot design Select the DNA Hotspot design option to design a panel to detect known SNPs and indels in your genes of interest. 1. Sign in to Ion AmpliSeq Designer with your Thermo Fisher Scientific login information. On the home page, click Made-to-Order Panels4Genes, Regions or Amplicons. Ion AmpliSeq Designer: Getting Started User Guide 31

32 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2. In the Start a new AmpliSeq design screen, in the Name and details step, enter a Design Name and optional Details in the appropriate fields. 3. Select DNA Hotspot designs (single-pool). Clicking the application type filters the compatible genomes shown. 4. Select a genome to use as reference. For custom references, click Custom Reference. 5. Click Next: Add Targets. 32 Ion AmpliSeq Designer: Getting Started User Guide

33 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2 6. Three options for adding hotspot targets are available: Hotspot by ID, Hotspot by Coordinates, and Upload File. Option Hotspot by ID Allows various manual options: Description 1. Enter the SNP ID, or start typing and select the full ID from the dropdown list. 2. 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. 3. When finished, click Submit targets. Hotspot by Coordinates Upload File Allows you to enter hotspot IDs assigned to specific genomic coordinates. Allows you to upload genomic coordinates of several targets at a time, via a SNP list or BED file (select from the "Type" dropdown list). Note: Click Input Specifications for more information. Ion AmpliSeq Designer uploads the targets, checks them, and verifies regions. 7. For any highlighted erroneous target(s), either correct the coordinates inside the table, or remove them by selecting their checkbox(es), then clicking Delete. 8. Select a DNA Type for the type of sample you plan to construct the library from: Standard DNA, FFPE DNA, or cfdna. Note: This setting affects amplicon length in the panel design. 9. (Optional) Click Export targets to download your targets into a CSV file. Ion AmpliSeq Designer: Getting Started User Guide 33

34 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 10. To submit your panel, click Submit targets. Note: Two designs can be submitted at a time. 11. Click OK to confirm your submission. When confirmed, you see a confirmation message and receive an confirming the design submission. 12. Proceed to step 13 in Start a new DNA Gene design to review and order your panel. 34 Ion AmpliSeq Designer: Getting Started User Guide

35 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2 Start a new RNA Gene Expression design Select the RNA Gene Expression design option to design a panel to measure differential expression of genes of interest in your sample types. 1. Sign in to Ion AmpliSeq Designer with your Thermo Fisher Scientific login information. On the home page, click Made-to-Order Panels4Genes, Regions or Amplicons. Ion AmpliSeq Designer: Getting Started User Guide 35

36 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2. In the Start a new AmpliSeq design screen, in the Name and details step, enter a Design Name and optional Details in the appropriate fields. 3. Select RNA Gene Expression designs (single-pool). Note: The RNA Gene Expression design pipeline is only compatible with the Human (RefSeq) reference. Clicking the application type filters the compatible genomes shown. 4. Click Next: Add Targets. 5. Two options for adding gene targets are available: Add Gene/Transcript, and Upload File. Option Add Gene/Transcript Upload File Allows manual options: Description 1. Start typing the gene symbol or RefSeq accession number, then select from the dropdown list 2. Click Add target after each entry. A green or red text box appears after each to indicate if the target was added successfully. 3. When finished, click Submit targets. Allows you to upload gene symbol or accession number list of many targets at a time, via a CSV or text file. 36 Ion AmpliSeq Designer: Getting Started User Guide

37 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2 Note: Click Input Specifications for more information. Ion AmpliSeq Designer uploads the targets, and checks them. 6. For any highlighted erroneous target(s), either correct the coordinates inside the table, or remove them by selecting their checkbox(es), then clicking Delete. 7. (Optional) Click Export targets to download your targets to a CSV file. 8. To submit your panel, click Submit targets. Note: Two designs can be submitted at a time. 9. Click OK to confirm your submission. When confirmed, you see a confirmation message and receive an confirming the design submission. 10. Proceed to step 13 in Start a new DNA Gene design to review and order your panel. Ion AmpliSeq Designer: Getting Started User Guide 37

38 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design Start a new RNA Gene Fusion design Select the RNA Gene Fusion design option to make an RNA Gene Fusion panel to detect expression of known gene fusions of interest in your samples. IMPORTANT! For successful panels, you must have at least 12 gene expression (GEX) assays per panel. We provide 12 default GEX assays for each panel that you can accept or replace with targets of your choice. For panels requiring two pools, the GEX assays are split between the two pools. 1. Sign in to Ion AmpliSeq Designer with your Thermo Fisher Scientific login information. On the home page, click Made-to-Order Panels4Genes, Regions or Amplicons. 2. In the Start a new AmpliSeq design screen, in the Name and details step, enter a Design Name and optional Details in the appropriate fields. Note: Human (RefSeq/Ensembl) is the only reference sequence that is allowed with this panel type. 38 Ion AmpliSeq Designer: Getting Started User Guide

39 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2 3. In the Application type step, select RNA Gene Fusion designs (multi-pool), then click Next: Add Targets. A screen appears for adding gene fusion targets to the panel. 4. In the Symbol Pair text box in the Add Fusion tab: type the gene symbol of the gene you want to add targets for. Select your targets from the dropdown list of available gene fusions that appears, or select Upload File to open a dialog, then browse to a file for upload. Note: Click Input Specifications for further information about adding fusion targets and symbol pair formats. Ion AmpliSeq Designer: Getting Started User Guide 39

40 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design After you select a fusion pair, available isoforms for the pair will appear in a table under the Symbol Pair field. 5. Add the fusions by selecting the checkbox in the row of each target, then click Add fusion(s). 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. 6. Repeat step 4 and step 5 to add additional fusion targets as needed. As you add fusion targets, the targets appear in a panel table at the bottom of the screen. Proceed to Add gene expression assays on page 41 to add or edit gene expression assays and finish the design. 40 Ion AmpliSeq Designer: Getting Started User Guide

41 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design 2 Add gene expression assays Every fusion panel is required to have 12 gene expression assays, and 12 are preselected by default to get you started. To pick your own gene expression assays, delete the default assays and add your own gene expression targets by gene symbol or RefSeq transcript accession. 1. Click the Add Genes/Transcript 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 Input Specifications 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. Note: 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 41

42 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel 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. 9. Review results in 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 in the Genes tab. 42 Ion AmpliSeq Designer: Getting Started User Guide

43 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Start a new Ion AmpliSeq Made-to-Order Panel design Click the Gene link to view compatible transcripts. Ion AmpliSeq Designer: Getting Started User Guide 43

44 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels 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, select Ready-to-Use Panels4DNA or RNA. A table displaying research panels and their descriptions appears. 44 Ion AmpliSeq Designer: Getting Started User Guide

45 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Browse Ion AmpliSeq Ready-to-Use and Community Panels 2 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 view more details by clicking More and Review Panel. Click Download panel files to obtain panel files required for analysis of your sequencing results. Ion AmpliSeq Designer: Getting Started User Guide 45

46 2 Chapter 2 Start a new Ion AmpliSeq panel design, or browse Ion AmpliSeq Ready-to-Use Panels Browse Ion AmpliSeq Ready-to-Use and Community Panels You can also browse pre-designed Community Panels in the same research areas by selecting Made-to-Order Panels4Pre-designed by our Community on the Ion AmpliSeq side of the home page. 46 Ion AmpliSeq Designer: Getting Started User Guide

47 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Gene design Ion AmpliSeq HD panels provide ultra-high sensitivity for your targeted sequencing applications. Select the DNA Gene design option to design a panel to discover new variants as well as detect known SNPs and indels in your genes of interest. 1. Sign in to Ion AmpliSeq Designer with your Thermo Fisher Scientific login information. On the home page, in the Ion AmpliSeq HD, Made-to-Order Panels pane (right side of screen), click Genes, Regions or Amplicons. Ion AmpliSeq Designer: Getting Started User Guide 47

48 3 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Gene design 2. In the Start a new AmpliSeq HD design screen, in the Name and details step, enter a Design Name and optional Details in the appropriate fields. 3. Select your Application type: DNA Gene designs (multi-pool). Note: Ion AmpliSeq HD panel designs are currently compatible only with the Human hg19 genome reference. Clicking the application type filters the compatible genomes that are shown. 4. Click Next: Add Targets. 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, then select the gene from the dropdown list. 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). 48 Ion AmpliSeq Designer: Getting Started User Guide

49 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Gene design 3 Note: Click Input Specifications for more information. Note: Ion AmpliSeq HD panel design numbers (shown at upper left) are designated with an "IAH" prefix. Standard Ion AmpliSeq panel designs are designated with an "IAD" prefix. Ion AmpliSeq Designer uploads the targets, checks them, and verifies regions. Ion AmpliSeq HD panel designs are limited to 500 amplicons per primer pool. Contact Thermo Fisher Scientific regarding Ion AmpliSeq HD panels that require more than two primer pools. 6. For any highlighted erroneous target(s), either correct the coordinates inside the table, or remove them by checking their checkbox(es), then clicking Delete. 7. Select an Exon padding value if different from the standard setting. 8. Select a DNA Type for the type of sample you plan to construct the library from: FFPE DNA, or cfdna. Note: This setting affects amplicon length in the panel design. 9. (Optional) Click Export targets to download your targets into a CSV file. 10. To submit your panel, click Submit targets. Note: Two designs can be submitted at a time. Ion AmpliSeq Designer: Getting Started User Guide 49

50 3 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Gene design 11. Click OK to confirm your submission. When confirmed, you see a confirmation message and receive an confirming the design submission. 12. 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). 50 Ion AmpliSeq Designer: Getting Started User Guide

51 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Gene design Use the following fields to review your order: Switch design Click the dropdown list 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 Chip Calculator Use the Chip calculator to obtain guidelines for sample library loading with various Ion chips to help in panel design. 6 Add to cart When this button is green you can 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. 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. Ion AmpliSeq Designer: Getting Started User Guide 51

52 3 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Hotspot design Start a new Ion AmpliSeq HD DNA Hotspot design Select the DNA Hotspot design option to design a panel to detect known SNPs and indels in your genes of interest. 1. Sign in to Ion AmpliSeq Designer with your Thermo Fisher Scientific login information. On the home page, in the Ion AmpliSeq HD, Made-to-Order Panels pane (right side of screen), click Genes, Regions or Amplicons. 52 Ion AmpliSeq Designer: Getting Started User Guide

53 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Hotspot design 3 2. In the Start a new AmpliSeq HD design screen, in the Name and details step, enter a Design Name and optional Details in the appropriate fields. 3. Select DNA Hotspot designs (single-pool). Note: Ion AmpliSeq HD panel designs are currently compatible only with the Human hg19 genome reference. Clicking the application type filters the compatible genomes shown. 4. Click Next: Add Targets. 5. Three options for adding hotspot targets are available: Hotspot by ID, Hotspot by Coordinates, and Upload File. Option Hotspot by ID Allows various manual options: Description 1. Enter the SNP ID, or start typing and select the full ID from the dropdown list. 2. 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. 3. When finished, click Submit targets. Hotspot by Coordinates Upload File Allows you to enter hotspot IDs assigned to specific genomic coordinates. Allows you to upload genomic coordinates of several targets at a time, via a SNP list or BED file (select from the "Type" dropdown list). Ion AmpliSeq Designer: Getting Started User Guide 53

54 3 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Hotspot design Note: Click Input Specifications for more information. Note: Ion AmpliSeq HD panel design numbers (shown at upper left) are designated with an "IAH" prefix. Standard Ion AmpliSeq panel designs are designated with an "IAD" prefix. Ion AmpliSeq Designer uploads the targets, checks them, and verifies regions. Ion AmpliSeq HD panel designs are limited to 500 amplicons per primer pool. 6. For any highlighted erroneous target(s), either correct the coordinates inside the table, or remove them by checking their checkbox(es), then clicking Delete. 7. Select a DNA Type for the type of sample you plan to construct the library from: FFPE DNA, or cfdna. Note: This setting affects amplicon length in the panel design. 8. (Optional) Click Export targets to download your targets into a CSV file. 9. To submit your panel, click Submit targets. Note: Two designs can be submitted at a time. 54 Ion AmpliSeq Designer: Getting Started User Guide

55 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD DNA Hotspot design Click OK to confirm your submission. When confirmed, you see a confirmation message and receive an confirming the design submission. 11. Proceed to step 12 in Start a new Ion AmpliSeq HD DNA Gene design on page 47 to review and order your panel. Ion AmpliSeq Designer: Getting Started User Guide 55

56 3 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD RNA Gene Fusion design Start a new Ion AmpliSeq HD RNA Gene Fusion design Select the RNA Gene Fusion design option to make an RNA Gene Fusion panel to detect expression of known gene fusions of interest in your samples. IMPORTANT! We provide 19 GEX assays for each Ion AmpliSeq HD RNA Gene Fusion panel that are fixed and uneditable. 1. Sign in to Ion AmpliSeq Designer with your Thermo Fisher Scientific login information. On the home page, in the Ion AmpliSeq HD, Made-to-Order Panels pane (right side of screen), click Genes, Regions or Amplicons. 56 Ion AmpliSeq Designer: Getting Started User Guide

57 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD RNA Gene Fusion design 3 2. In the Start a new Ion AmpliSeq HD design screen, in the Name and details step, enter a Design Name and optional Details in the appropriate fields. Note: Human (RefSeq/Ensembl) is the only reference sequence that is allowed with this panel type. 3. In the Application type step, select RNA Gene Fusion designs (single-pool), then click Next: Add Targets. A screen appears for adding gene fusion targets to the panel. Ion AmpliSeq Designer: Getting Started User Guide 57

58 3 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD RNA Gene Fusion design Note: Ion AmpliSeq HD panel design numbers (shown at upper left) are designated with an "IAH" prefix. Standard Ion AmpliSeq panel designs are designated with an "IAD" prefix. Gene expression controls for Ion AmpliSeq HD are fixed and not editable, unlike Ion AmpliSeq Made-to-Order fusion designs. 4. In the Symbol Pair text box in the Add Fusion tab, type the gene symbol of the gene you want to add targets for. Select your targets from the dropdown list of available gene fusions that appears. Alternatively, select Upload File to open a dialog, then browse to a file for upload. Note: Click Input Specifications for further information about adding fusion targets and symbol pair formats. After you select a fusion pair, available isoforms for the pair will appear in a table under the Symbol Pair field. 58 Ion AmpliSeq Designer: Getting Started User Guide

59 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD RNA Gene Fusion design 3 5. Add the fusions by selecting the checkbox in the row of each target, then click Add fusion(s). 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. 6. Repeat step 4 and step 5 to add additional fusion targets as needed. As you add fusion targets, the targets appear in a panel table at the bottom of the screen. 7. To submit your panel, click Submit targets. Note: Two designs can be submitted at a time. Ion AmpliSeq Designer: Getting Started User Guide 59

60 3 Chapter 3 Start a new Ion AmpliSeq HD panel design Start a new Ion AmpliSeq HD RNA Gene Fusion design 8. Click OK to confirm your submission. When confirmed, you see a confirmation message and receive an confirming the design submission. 9. Proceed to step 12 in Start a new Ion AmpliSeq HD DNA Gene design to review and order your panel. 60 Ion AmpliSeq Designer: Getting Started User Guide

61 A Troubleshooting For complete Ion AmpliSeq library troubleshooting, see the following user guides, available at thermofisher.com: Ion AmpliSeq Library Kit 2.0 User Guide (Pub. No. MAN ) Ion AmpliSeq Library Kit Plus User Guide (Pub. No. MAN ) Ion AmpliSeq HD Library Kit User Guide (Pub. No. MAN ) Ion AmpliSeq Designer: Getting Started User Guide 61

62 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 Ion Chip capacities for Ion AmpliSeq DNA libraries sequenced at equal depths Ion Chip capacities for Ion AmpliSeq RNA libraries 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 custom 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 dbsnp identifiers, use the UCSC Table Browser of the UCSC Genome Browser. Make sure to select genome and assembly as Human, (GRCh37/hg19). 62 Ion AmpliSeq Designer: Getting Started User Guide

63 Appendix B Supplemental information Padding coordinates or create a BED file from list of variants B Start from a list of dbsnp target identifiers Use the following example of creating a BED file from dbsnp targets with the UCSC Table Browser to generate BED files from other variant formats, such as ClinVar. 1. Select the Variation group in the group dropdown list. 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 either the paste list or upload list button next to identifiers. 3. Either upload a text file listing your dbsnp identifiers or simply paste in the list, then click submit. For the output format, select BED - browser extensible data. Ion AmpliSeq Designer: Getting Started User Guide 63

64 B Appendix B Supplemental information Padding coordinates or create a BED file from list of variants 4. After clicking 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 Ion AmpliSeq Designer. 64 Ion AmpliSeq Designer: Getting Started User Guide

65 Appendix B Supplemental information Padding coordinates or create a BED file from list of variants B 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: UCSC RefSeq (refgene) Output format: BED - browser extensible data 1. Upload or paste a list of gene identifiers. After you click submit, you are 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. Select the appropriate options to create the BED file to include 5' or 3' UTR Exons, or only coding exons. Ion AmpliSeq Designer: Getting Started User Guide 65

66 B Appendix B Supplemental information Reference FASTA sequence 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 (for example, SNPs, indels, or other variations). Ion AmpliSeq Designer minimizes primer overlap with these regions. This file is. You can upload it at the time of creating a new custom reference. The BED format is a tab-delimited file, with one line per region. Required fields are chrom, chromstart, and chromend in the first three columns of the BED file format. Additional fields are ignored. The chrom field must match one contig ID in the accompanying FASTA file. chromstart and chromend fields are the zero-based, half-open coordinates 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. 66 Ion AmpliSeq Designer: Getting Started User Guide

67 Appendix B Supplemental information Pipeline details B The chrom, chromstart, and chromend fields must meet the following criteria: chromstart can be a value between 0 and length of the sequence specified by chrom 1. chromend must be greater than chromstart. chromend can have a maximum value of the length of the sequence specified by chrom. No region should overlap any other region in the file. Combine overlapping regions into a single contiguous region. The variant coordinates BED file must have no header (for example, no "track" lines). Example FASTA file (50 bases per line): Sample variants of interest (highlighted in blue). 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. Ion AmpliSeq Designer: Getting Started User Guide 67

68 B Appendix B Supplemental information Pipeline details 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. 68 Ion AmpliSeq Designer: Getting Started User Guide

69 Appendix B Supplemental information Hotspots pipeline 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 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. Ion AmpliSeq Designer: Getting Started User Guide 69

70 B Appendix B Supplemental information Hotspots pipeline 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. 70 Ion AmpliSeq Designer: Getting Started User Guide

71 Appendix B Supplemental information Ion Chip capacities for Ion AmpliSeq DNA libraries sequenced at equal depths B Ion Chip capacities for Ion AmpliSeq DNA libraries sequenced at equal depths The number of combined libraries that can be accommodated in a single sequencing run depends on the chip, the balance of barcoded library concentration, and the coverage required. For a given chip, as the number of amplicons increases, the number of libraries that can be accommodated per sequencing run decreases. This relationship is shown in the following table. The numbers in the following table serve as a guide for approximate capacities. As the number of libraries per chip increases, it becomes more difficult to balance the reads between libraries. In addition, libraries from FFPE tissue tend to produce more variable results. We suggest combining fewer libraries initially and determining real limits empirically. Chip Ion 314 Ion 316 Ion 510 Ion 318 Ion 520 Ion 530 Ion PI Ion 540 Ion 550 Average depth 150X 2500X 150X 2500X 150X 2500X 150X 2500X 150X 2500X 150X 2500X Expected coverage 95% >30X 95% >500X 95% >30X 95% >500X 95% >30X 95% >500X 95% >30X 95% >500X 95% >30X 95% >500X 95% >30X 95% >500X Number of amplicons per library Approximate number of libraries per chip > > >384 >384 >384 > > > >384 > > > > > > > > > , ,537 3, ,073 6, ,145 12, ,289 24, ,577 49, Ion AmpliSeq Designer: Getting Started User Guide 71

72 B Appendix B Supplemental information Ion Chip capacities for Ion AmpliSeq RNA libraries Ion Chip capacities for Ion AmpliSeq RNA libraries We recommend that you plan for an average of 5,000 reads per amplicon for an Ion AmpliSeq RNA library targeting genes. The actual sequencing depth that is required depends on the expression levels of the gene targets in your sample RNA, so scale the sequencing depth to accommodate your sample type and research needs. For panels containing fusion detection primer pairs, higher library multiplexing is possible as most targets will not be present, and therefore will not create library molecules. For most fusion detection assays, only ~250,000 reads/library are required. Use the following formula and chip capacity table to provide initial guidance for multiplexing RNA-derived gene expression libraries on Ion sequencing chips. Number of libraries sequenced per chip = (Sequencing depth) Chip capacity in reads (Number of primer pairs) These recommendations serve as suggestions only and the actual capacity to multiplex libraries is determined by the expression levels of the genes included in your Ion AmpliSeq RNA panel. The expression levels of the individual genes can vary by input RNA type as well. We suggest using the formula for new panels and determining actual multiplexing limits empirically. Ion Chip Ion 314 Chip Ion 316 Chip Ion 510 Chip Ion 318 / Ion 520 Chip Ion 530 Chip Ion PI / Ion 540 Chip Ion 550 Chip Chip capacity in reads (M) Example: Chip capacity of Ion 540 Chip = ~60,000,000 reads Sequencing depth desired = 5000 reads per amplicon Number of primer pairs = ,000,000 / (5, ) = 120 libraries per Ion 540 Chip 72 Ion AmpliSeq Designer: Getting Started User Guide

73 Appendix B Supplemental information Frequently asked questions (FAQs) B 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. Ion AmpliSeq Designer: Getting Started User Guide 73

74 B Appendix B Supplemental information Frequently asked questions (FAQs) 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/ Ion AmpliSeq Designer: Getting Started User Guide

75 Appendix B Supplemental information Frequently asked questions (FAQs) B Ion AmpliSeq On Demand Panels FAQs General Ion AmpliSeq On-Demand Panel design questions 1. Why can I order only 500 genes in an On-Demand panel? For this version of the software, we have set an ordering limit to 500 genes or 15,000 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 500 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. Ion AmpliSeq Designer: Getting Started User Guide 75

76 B Appendix B Supplemental information Frequently asked questions (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 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. 76 Ion AmpliSeq Designer: Getting Started User Guide

77 Appendix B Supplemental information Frequently asked questions (FAQs) B 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 (ampliseqdesigns@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. Ion AmpliSeq Designer: Getting Started User Guide 77

78 B Appendix B Supplemental information Frequently asked questions (FAQs) 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. 78 Ion AmpliSeq Designer: Getting Started User Guide

79 Appendix B Supplemental information Frequently asked questions (FAQs) B Ion AmpliSeq HD FAQs 1. When should I use Ion AmpliSeq HD (ASHD) and not "regular" Ion AmpliSeq (AS)? It depends on the application. If you have an application that requires ultra-high sensitivity, then ASHD is recommended. If you have applications that require high multiplexing, then regular AS is the preferred option. 2. What is the maximum number of amplicons per pool that are supported by ASHD? ASHD currently supports up to 500 amplicons per pool with a 2-pool maximum of 1,000 amplicons total. This limit is enforced in AmpliSeq.com. 3. How many reactions worth of material can I expect in my order of an ASHD panel? The approximate number of reactions worth of material provided for ASHD panels is 3,000 reactions. 4. Designs are only available for cfdna ( bp amplicons) and FFPE ( bp amplicons), what about Germline applications ( bp and bp amplicons)? Germline applications may be considered in the future, depending on the relevance of use of ASHD technology. 5. What are the variant types supported by ASHD? The current supported variant types are, SNVs, small indels, fusions from a predesigned list, and CNV through design best practices available in AmpliSeq.com. 6. What happens if my desired fusion is not available in AmpliSeq.com? In this case, a request to the AmpliSeq Custom Services team should be submitted. Contact your local sales or support representative to learn more. 7. Does the AmpliSeq Custom Services team support specialty designs for ASHD? Yes, the AmpliSeq Custom Services team is supporting specialty designs for ASHD. Submission of specialty ASHD designs to the AmpliSeq Custom Services team should follow the same path for AS design requests, which involves contacting your sales or field support contacts for help. 8. Only gene expression controls are available when creating a fusion design, what about custom designs for gene expression assays, are these supported by ASHD? Automated gene expression designs are not currently supported in AmpliSeq.com. However, custom gene expression designs can be created by the AmpliSeq Custom Services team. Contact your local sales or support representative to learn more. 9. The new Oncomine cf PanCancer panel contains DNA and RNA targets in 1- pool, can you create a similar design with ASHD? Yes, but only through our AmpliSeq Custom Services team. Designs that are created in AmpliSeq.com are limited to 1-pool for DNA hotspots, 1-pool for RNA fusions, and 2-pools for DNA gene designs. 10. Why are the FWD and REV primers kept in separate pools? The primers used in ASHD are more complex than for regular AS, so it has been recommended to keep them in separate pools for storage and long term stability. FWD and REV primers should only be mixed at the time the libraries are created. See the Ion AmpliSeq HD Library Kit User Guide (Pub. No. MAN ) for more information. Ion AmpliSeq Designer: Getting Started User Guide 79

80 B Appendix B Supplemental information Related Ion sequencing products Related Ion sequencing products Link to product description page Ion AmpliSeq Library Kit 2.0 Ion AmpliSeq Library Kit Plus Ion AmpliSeq HD Library Kit Ion AmpliSeq Sample ID Panel Ion Library Equalizer Kit Ion Xpress Barcode Adapters 1 16 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. Ion AmpliSeq HD DNA and RNA dualbarcoded library preparation from targeted DNA and RNA 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, Ion S5 /Ion S5 XL, and Ion GeneStudio S5 Series sequencers. When used in combination with the Ion AmpliSeq Library Kit 2.0 or the Ion AmpliSeq Library Kit Plus, 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. 80 Ion AmpliSeq Designer: Getting Started User Guide

81 Appendix B Supplemental information Related Ion sequencing products B Link to product description page IonCode Barcode Adapters Kit Description The IonCode Barcode Adapters Kit provides 384 different pre-mixed adapters in a convenient 96-well plate format. Ion AmpliSeq HD Dual Barcode Kit 1 24 The Ion AmpliSeq HD Dual Barcode Kit 1 24 provides 24 dual barcode primers for Ion AmpliSeq HD library preparation. Ion AmpliSeq Kit for Chef DL8 Ion PGM Hi Q View Chef Kit Ion PI Hi Q Chef Kit Ion 510 & Ion 520 & Ion 530 Kit Chef Ion 540 Kit Chef Ion 550 Kit Chef Ion 314 Chip Kit v2 BC Ion 316 Chip Kit v2 BC Ion 318 Chip Kit v2 BC Ion PI Chip Kit v3 Ion 510 Chip Kit Ion 520 Chip Kit Ion 530 Chip Kit Ion 540 Chip Kit Ion 550 Chip Kit 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, Ion S5 XL, and Ion GeneStudio S5 Series sequencers. Ion Chip kits contain chips for sequencing runs on the Ion PGM, Ion Proton, Ion S5, Ion S5 XL, and Ion GeneStudio S5 Series 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. Ion AmpliSeq Designer: Getting Started User Guide 81

82 B Appendix B Supplemental information Helpful tools 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. 82 Ion AmpliSeq Designer: Getting Started User Guide

83 Appendix B Supplemental information Advanced features and tools B 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 Ion AmpliSeq Designer 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. Ion AmpliSeq Designer: Getting Started User Guide 83

84 B Appendix B Supplemental information Advanced features and tools 2. Copy the sequence, and run a BLAST search on this sequence. 3. Select Human, running a nucleotide query. 84 Ion AmpliSeq Designer: Getting Started User Guide

85 Appendix B Supplemental information Advanced features and tools B 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 Percent track options. Ion AmpliSeq Designer: Getting Started User Guide 85

86 B Appendix B Supplemental information Advanced features and tools 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 Torrent Suite Software to determine coverage uniformity of your sequence run. For further information on using the coverageanalysis plugin, see Torrent Suite Software 5.10 Help. 86 Ion AmpliSeq Designer: Getting Started User Guide