Frequently Asked Questions


Select an FAQ Topic to Explore:

Ion Torrent Genexus System - Consumables

  1. When selecting the Genexus instrument, I can’t find the Special Instructions option, why?

    Special Instructions are only supported when selecting the GeneStudio or PGM/Proton Instrument at this time.

  2. Why can’t I select additional consumables when selecting the PGM/Proton instrument option?

    The option for selecting additional consumables is only available for the Genexus or the GeneStudio instruments. If you need to select additional consumables for PGM/Proton, please visit the thermofisher.com website directly.

  3. Are designs created from references other than the hg19 genome reference supported in Genexus?

    Unfortunately, not at this time. Only designs created using the hg19 reference are supported for use with the Genexus Instrument.

  4. Is there an easy way for users to understand which products are supported for which instruments?

    Yes, please refer to the following table:

    Products: Instrument
    Genexus GeneStudio PGM Proton
    AmpliSeq Made-to-Order DNA (hg19)
    AmpliSeq Made-to-Order (other genomes) DNA
    AmpliSeq Made-to-Order RNA (RefSeq)
    AmpliSeq HD DNA (hg19)
    AmpliSeq HD RNA (RefSeq)
    AmpliSeq On-Demand
    Oncomine tumor specific panels
  5. Are all Community panels supported for all journeys?

    Only Community panels for DNA are supported in the Genexus System. All other Community panels are only supported on the GeneStudio and PGM/Proton instruments.

  6. Are panels created by the AmpliSeq Custom Services team (White Glove) supported for all instruments?

    If the design is created using the hg19 reference, then it is supported for all instruments, otherwise, the designs are only supported for the GeneStudio or PGM/Proton instruments.

  7. When selecting my Read-to-Use panels, I don’t get the option to select additional consumables in ampliseq.com, why?

    When ordering a Ready-to-Use panel, the user is sent to the appropriate product page on thermofisher.com, which already contains additional consumables suggested for the panel.

  8. In the additional consumables page, why can I only edit certain parameters?

    Parameter values are inherited from the design, for example, number of amplicons or number of pools, and cannot be changed. Only parameters affecting the selection of the consumables, are available for editing.

  9. Is there documentation available that can help me understand the relationship between samples per chip versus samples per lane for the Genexus GX5 chip?

    Please refer to the Genexus User Guide (page 136) for more information regarding how many samples can be run on the GX5 chip.

Oncomine tumor specific panels – General Panel design

  1. What sample types are compatible?

    Oncomine tumor specific panels were designed to be compatible with degraded DNA such as FFPE, but can also be used on FNA (fine needle aspirate), fresh frozen samples, and other high quality DNA.

  2. What is the price? How is pricing determined?

    Pricing is based on the panel configuration: Manual vs Chef library preparation, reaction pack size and number of genes in the panel.

    Reactions core Customized
    Manual Chef Manual Chef
    24 96 384 32 128 24 96 384 32 128
    1-10 genes
    11-30 genes
    31-50 genes
    51-75 genes
    76-100 genes
    101-150 genes
  3. I don’t see Oncomine Tumor Specific panels in the chip calculator. How many samples per chip?

    The 530 chip is supported; in addition, estimates are provided for other chips below, based on the number of genes in the DNA panel:

    Chip GeneStudio PGM Genexus
    510 520 530 540 550 316 318 GX5
    max 3,000,000 5,000,000 20,000,000 80,000,000 130,000,000 3,000,000 5,500,000 60,000,000
    min 2,000,000 3,000,000 15,000,000 60,000,000 100,000,000 2,000,000 4,000,000 48,000,000
    avg 2,500,000 4,000,000 17,500,000 70,000,000 115,000,000 2,500,000 4,750,000 54,000,000
    1-10 genes 10 2 4 20 81 133 2 5 62
    11-30 genes 30 - 1 6 27 44 - 1 20
    31-50 genes 50 - - 4 16 26 - 1 12
    51-75 genes 75 - - 2 10 17 - - 8
    76-100 genes 100 - - 2 8 13 - - 6
    101-150 genes 150 - - 1 5 8 - - 4

    * Assumes 43 amplicons/gene, max number of genes per pricing tier and 2000x depth.

Oncomine tumor specific panels – Panels and Panel Content

  1. Being Oncomine branded, can you do lot matching?

    You should expect each order of a panel to be a new lot number. However, there is a possibility that 2 orders provided on the same SO could be the same lot number.

  2. I see some tumor types are missing, will these get added eventually, like brain or sarcoma?

    We are investigating the addition of new tumor types and new genes in the future. Please contact support or provide assay feedback on AmpliSeq.com for any additions you’d like to see, including additional genes. Specific design feedback should be provided using the Give Solution Feedback option in the More Actions drop-down menu on the panel design page. To provide general feedback or request help, use the Assay Design Support link in the footer of each page on Ampliseq.com.

  3. What is the difference between a core panel and customized panel?

    All genes in inventory have been tested together to assure they perform well together in any permutation, core or customized. The core panels (tumor specific panels) and genes have been tested to the highest quality requirement compared to other genes in inventory as these will be most relevant to clinical research. In addition, each core panel has had rigorous testing on FFPE samples and positive controls to set realistic expectations of panel performance.

  4. What is the maximum and minimum number of genes/amplicons allowed?

    The minimum number of amplicons is 24 (12 per pool). The maximum number of amplicons is 5000 or 150 genes, whichever is reached first.

  5. The added library kits have different product numbers than I’m used to. Are they different than the ones available on Thermofisher.com?

    The library kits are the same as those commercially available, simply under a different product number. These library kits are only available with the Oncomine tumor specific panels from Ampliseq.com.

  6. What rationale can you provide for the core panel gene content? What are associated research genes?

    Core panel content is designed to be most relevant for clinical research of the tumor type based on drug labels, guidelines and clinical trials. The panel can be customized if you prefer to add or replace certain genes.

    Associated research genes may also be of interest based on our curation of the relevant literature.

  7. Can you differentiate the B cell vs T cell origin with the Lymphoma panel?

    Not currently. When a future panel includes targets for both cell types, you will be able to determine the cell type of origin.

  8. Why are the genes that I expect to see on the lymphoma panel not there?

    We have omitted all the fusion (RNA) genes from this panel design. Support for fusions will be added in a future release. Even if there is a relevant (DNA) mutation on one of the fusion genes, it is omitted, so that one does not assume that the RNA component is included in the panel design. For instance, ALK fusions are relevant, as are ALK mutations. ALK is omitted from this version of the panel design.

Oncomine tumor specific panels – Designer: AmpliSeq.com

  1. What does the hotspot track indicate in the IGV viewer?

    Hotspots are regions in the genome where the risk for biologically relevant genetic mutations is elevated (eg. InDels and SNPs). Internally we have curated a list of relevant hotspots that are annotated in Ion Reporter. Amplicons were designed to optimally cover these hotspots. Some genes have amplicons covering the entire gene: other genes have amplicons designed to cover all relevant hotspots, but not necessarily the entire gene. The hotspots are displayed in the new track on AmplISeq.com, with expected amplicon coverage.

  2. Where is the spike-in feature of AmpliSeq On Demand? What do I do if my gene isn’t present?

    We do not currently offer a spike-in function for the Oncomine tumor specific panels. All genes have been designed with respect to each other in order to enable high performance panels, core or customized. We cannot guarantee performance of the panel when a MTO spike-in is used. If your gene is not present, please contact support and we will attempt to include it in the next phase of genes. An assay feedback tool is available on the designer – please submit requests to add new genes to inventory.

  3. What if I just want amplicons to cover hotspots for a certain gene, not the full gene design?

    Each gene is available as is without modification. Please contact support for help designing amplicons covering only your genomic location of interest for inclusion into a spike-in. An assay feedback tool is available on the designer – please submit any requests for modification to a gene design.

  4. What does design finalization mean?

    Design finalization involves generation of all of the final panel files. Some designs may take longer than 30 minutes. Contact support if the process is not complete within a couple of hours.

Oncomine tumor specific panels – Panel Design and Performance

  1. What’s the quality guarantee?

    Each gene has been tested on FFPE samples in the context of all inventoried genes, and in the context of tumor-specific panels where appropriate, to ensure performance in any panel environment. However, since full customization is possible, not every possible panel permutation will be tested, and thus some gene and amplicon variability should be expected. Each tumor-specific panel has had additional testing. We strive to achieve a high product quality to provide you with consistent and reliable panel performance, please contact us if you have questions or concerns.

  2. Why does this product have an Assessment package and Development packages but other Oncomine panels don’t? Will they in the future?

    As a custom product, each panel order is considered a new lot. We recommend testing each new lot to make sure it meets expectations. To support this effort, we are providing an assessment package which includes normal genomic DNA and FFPE DNA to assess panel coverage and sequencing depth uniformity as well as a positive control to assess preliminary performance. We have heard the need for the ability to customize an Oncomine panel and/or run an Oncomine panel on a different chip and maintain confidence in panel performance. We have responded with the Development Package. This package contains sufficient normal genomic DNA and FFPE DNA for SVB and CNB generation, respectively, as well as a positive control to assess CNV calling.

Oncomine tumor specific panels – Analysis and Workflow

  1. Can I detect copy number changes? Which genes?

    All genes, whether CDS or hotspot, have been designed with sufficient amplicons to enable CNV detection. BTK, a hotspot only gene, is the only exception.

  2. What is the supported workflow?

    The tumor-specific panels are supported on the 530 chip on the Ion GeneStudio instruments. Manual library preparation is supported with the Ion AmpliSeq Library kit Plus and dual barcodes. Chef library preparation is supported with the Ion AmpliSeq for Chef DL8 kit and IonCode barcodes. Dedicated analysis workflows for the tumor-specific panels are available in Ion Reporter. Generation of Reports is supported in Oncomine Reporter.

  3. Can I run a tumor specific panel on a non-530 chip? What do I need to make sure I can call SNPs/InDels? What about CNVs?

    Yes, you can run a tumor specific panel on a non-530 chip. You will need to create a custom CNV baseline, SVB (sequence variant baseline) and hotspot file and upload to a custom Ion Reporter analysis workflow. See the Oncomine tumor specific panel User Guide (MAN0018937: Ion Torrent Oncomine tumor specific panels) for the detailed protocol and ask your FAS/FBS rep for support if needed.

  4. Can I run a customized panel on a 530 chip? What do I need to make sure I can call SNPs/Indels properly? What about CNVs?

    Yes, you can run a customized tumor specific panel on a 530 chip. You will need to make a custom Ion Reporter analysis workflow using the BED file from AmpliSeq.com as well as create a custom CNV baseline. See the Oncomine tumor specific panel User Guide (MAN0018937: Ion Torrent Oncomine tumor specific panels) for the detailed protocol and ask your FAS/FBS rep for support if needed.

  5. When I run CNV analysis in IR, there is CNV detected at chr3:193207277 that isn’t associated with a gene. Is this real?

    Oncomine tumor specific assay analysis results may indicate a CNV at chr3:193207277 of 116bp in length and not associated with a gene. It is not a CNV hotspot but a sample ID amplicon and should be disregarded.

  6. How do I use the Oncomine BRCA workflow in IR to detect exon deletions in BRCA1 and BRCA2?

    You can detect exon deletions for BRCA1 in the Oncomine BRCA Expanded panel, or a customized panel thereof, by analyzing your sample with the Oncomine BRCA workflow in Ion Reporter 5.10 or 5.12. You should first run the IR Oncomine BRCA Extended workflow to detect SNPs, Indels, and gene level CNVs in all the genes in your panel. After that, you should run the IR Oncomine BRCA workflow to examine the exon deletions in BRCA1 and BRCA2. We are working to integrate the two workflows into a single workflow in a future version of Ion Reporter, to launch after IR 5.12. Note, CNV baselines can always be augmented with your own samples to increase the number of samples passing QC.

    a) Why are some of the variants detected in BRCA1 and BRCA2 in the IR Oncomine BRCA workflow different than those detected in the IR Oncomine BRCA Extended workflow?

    The Oncomine BRCA Expanded workflow is optimized to the tumor specific panel and includes analysis of all the genes in the panel. In contrast, the Oncomine BRCA workflow only analyses BRCA1 and BRCA2.

    b) Why does the on-target look low when I use the Oncomine BRCA workflow?

    The Oncomine BRCA workflow only analyses BRCA1 and BRCA2, thus the on-target reported is artificially low because it ignores all the amplicons that come from the other genes in the Oncomine BRCA Expanded panel or your custom panel. Use the on-target number reported in the Oncomine BRCA Expanded workflow, or your custom workflow.

    c) Why are the rest of the genes in the panel missing when I use the Oncomine BRCA workflow?

    The Oncomine BRCA Expanded workflow is optimized to the tumor specific panel and includes analysis of all the genes in the panel. In contrast, the Oncomine BRCA workflow only analyses BRCA1 and BRCA2 and only shows SNPs, Indels and CNVs in those two genes.

Ion AmpliSeq Designer - Design Sharing

  1. Why did the design sharing functionality change?

    The design sharing functionality has been enhanced to provide our users with better oversight and control of which designs have been shared and who they’ve been shared with.

  2. Does my collaborator still have access to my previously shared designs?

    No, the previously shared design links will no longer work. For collaborators to have access to previously shared designs, the collaborator will need to request access from the creator of the design. The design author will need to re-share their previously shared design using the new sharing functionality.

  3. How do I manage my shared designs?

    In the user dashboard, new icons and filtering options have been added to help users manage their shared designs. These icons provide with a view of the collaborators without having to open the design view.

  4. How can I share a design to multiple collaborators at once?

    Our current implementation only allows for one design to be shared at once. If you need to share the same design with multiple collaborators, you will need to share the design with each collaborator one at a time.

  5. How do I stop sharing a design with a collaborator?

    In order to stop sharing a design, you will need to visit the “Sharing” functionality and click the waste bin icon. This action will remove the selected user from having access to the design.

  6. What happens if I delete a shared design by mistake?

    Whether it is done intentionally or unintentionally, when you delete a design your collaborators will no longer have access to the design. Contact our support team if you have unintentionally deleted a design.

  7. Why is the email address of my collaborator not accepted?

    At this time only email addresses that have been registered in www.ampliseq.com are recognized as email valid emails. If you would like to share a design to your collaborator, encourage them to visit www.ampliseq.com and register. Before the design has been shared, they will need to log in to the site and accept our Terms and Conditions.

Ion AmpliSeq HD – General Panel Design

  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 5000 amplicons per pool with a 2-pool maximum of 10,000 amplicons total. This limit is enforced in AmpliSeq.com.

  3. How many reactions worth of material can I expect in my order of an 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 and FFPE, what about Germline applications?

    Germline applications may be considered in the future, depending on the relevance use of ASHD technology.

  5. What are the variant types supported by ASHD?

    The currently supported variant types are, SNVs, small Indels, Fusions from a pre-designed 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 will need to be submitted. Contact your local sales or support representative to learn more.

  7. Will the AmpliSeq Custom Services team support specialty designs for ASHD?

    Yes, the AmpliSeq Custom Services team will be 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 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. Please refer to ASHD Library Kit User Guide for more information.

Ion AmpliSeq HD – Oligo Ordering

  1. How many reactions do I receive when I order my DNA/RNA AmpliSeq HD design in Tubes Only format?

    Refer to the link for a table describing the number of reactions that you can expect, depending on the size of your panel.

  2. How many reactions do I receive when I order my DNA/RNA AmpliSeq HD design in Plates Only format?

    Refer to the link for a table describing the number of reactions that you can expect, depending on the size of your panel.

  3. AmpliSeq Made-to-Order offers the option for Tubes and 384-well plates, is this option available for AmpliSeq HD?

    The only order options available are, either Tubes Only or Plates Only option. If you need to order Tubes and Plates, we recommend placing 2 separate orders.

Ion AmpliSeq On-Demand – General Panel Design

  1. Why can I only order 500 genes in my panel?

    For this version of the software, we’ve set an ordering limit to 500 genes or 15,000 amplicons per panel due to manufacturing restrictions.

  2. Why is there a limit on the number of genes that I can add to my panel?

    Since 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 will need to be deselected in order to make the design orderable. For this reason, we’ve introduced a limit on the number of genes that can be added to an On-Demand panel.

  3. Can I edit the content once I’ve created a design?

    Yes, an On-Demand design can be edited after it has been created as long as it has not been ordered, added to cart 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 once the job has been submitted and the Results reported. If the On-Demand panel is locked, the panel can be cloned (or copied) to be unlocked and edited under a new IAD.

  4. Can I download my list of targets once I’ve created a design?

    Yes, select the “Export targets” button to download the list as a CSV file. This will export all the selected targets displayed in the user interface.

  5. Once I have created a design, can I add more content from a Disease Research Area (DRA)?

    Not directly. Currently, we do not allow the addition of DRA content or hierarchy levels to an existing design, only when the design is being created. The solution is to create a new design with the desired DRA content or hierarchy levels and add content from inventory as desired when in the unlocked design state.

  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 selected (green), will be ordered. If you create a Spike-in panel, that panel needs to be submitted and ordered separately by visiting the results page of that panel. The Spike-In panel is processed as a made-to-order custom panel and follows the same design submission and ordering process, including separate manufacturing and shipping.

  7. Can I edit my design once I’ve placed an order?

    No, once you’ve 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 to copy the panel design. A new IAD number will then be assigned to your design, and you will have the option to edit the design content. Cloning a panel will copy the entire design, selecting any available genes from inventory including any from the Spike-In panel that is now available from potential inventory updates.

  8. Can I reorder a design once I’ve placed an initial order?

    Yes, you can always go back to your ordered design and place a new order. If you want to order multiple copies of a panel, the solution is to either order a larger reaction size of the panel (ie one 96 rxn vs three 24rxn) or to go back to your design and add to cart again.

  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’re using is version v74.

  10. Are untranslated regions (UTRs) included with a gene design?

    No, only the coding DNA sequence (CDS) region of a gene is included as part of an On-Demand gene design. If UTRs or amplicons are desired, please contact the support team for potential spike-in solutions via Made-to-Order pipeline.

  11. Are UTR-only genes supported? What about pseudogenes?

    No, only genes containing CDS regions are supported. At this time, pseudogenes are not supported.

  12. What is the padding used for gene designs?

    The padding for every On-Demand gene design is either 5 bp or 25 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 (ie custom) design?

    We currently do not support a simple sharing mechanism. 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. We have wet-lab tested all inventoried content in-house.

  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 tested all possible gene combinations 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 our in-house R&D team has done is use computer-based searches to reduce as much as possible the occurrence of primer-primer interactions. The risk is not negligible but deemed very low, backed further by the number of satisfied customers. We have observed << 1% amplicon drop-out due to suspected primer-primer interactions.

    Further, we cannot guarantee specifications regarding off-target. We support the in-house GBU and coverage indicated in the IGV viewer and will do our best help troubleshoot if there are any issues that we believe are due to the design or manufacture or our panels.

  17. If my design has a banner indicating a gene(s) has amplicons greater than designed 325bp, what should I do?

    The default sequencing protocol for On Demand panels is 200bp and 550 nucleotide flows on any chip. If the user wants end to end reads on amplicons greater than 325bp (which can increase detection and accuracy of variant calls by reading both strands), we recommend increasing the number of flows to 650 and using 510, 520 or 530 chips. Amplicons between 275bp and 325bp can use the default workflow. Note, with a greater number of flows than the default, only one run per initialization will be possible. The user can determine the size of the insert from the bed file and then identify the amplicons greater than 325bp by adding on the length of the adapters/barcodes (~48 bp).

  18. What is the shelf life for these panels? How should they be shipped/stored? Do you supply a COA?

    Shelf life: 730 days based on the gene with the earliest manufacturing date.

    Shipped: RT. Stored at -20 deg C for longest stability. Avoid freeze-thaw cycles.

    COA: As this is a custom panel we do not have a standard COA are per kitted and SKUs off-the-shelf products. We can make a COA upon request stating stability of the oligos for a specific design.

  19. What is the turnaround for these panels once I place an order?

    As these are custom panels, made upon receipt of the order, the turnaround times can vary depending on geographical location but we aim for 2-3 weeks. We have no guarantees for TAT.

  20. How do I scale up my panel?

    We currently do not offer On-Demand panels in larger reactions packs than 32 rxn for Chef or 96 rxn for manual.

Ion AmpliSeq On-Demand – Disease Research Areas (DRAs)

  1. What are the sources used for creating the associations for the various Disease Research Areas found in the tool?

    The sources include DisGeNET , Unified Medical Language System and Medical Subject Headings (MeSH).

  2. What algorithm was used to create the Disease Research Areas gene-disease associations?

    An in-house gene scoring algorithm was used to create these associations. Details of the algorithm are proprietary but have been described at various national conferences. Our white paper can be found here . (link to be provided)

  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. The algorithm to determine scoring is proprietary.

  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 in order 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 on 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 the number of genes in that hierarchical level.

  7. The gene count doesn’t seem to add up. Why is that?

    Gene counts often don’t add up as the sum of the subcomponents because one or more genes can belong to multiple DRA’s.

  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, outside of the sources we used or did not meet our strict in-silico or wet-lab testing specifications. Contact our support team (ampliseq-designs@thermofisher.com) if you are aware of strong evidence demonstrating that a gene should be included in a specific category.

    Likewise, if you have any feature requests please don't hesitate to reach out to your rep or our support team (ampliseq-designs@thermofisher.com) and we can prioritize in future software releases.

  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.

  11. I want to create a panel for inherited oncology or CGx. Where is this in the DRA?

    Under "Neoplasms", you will see a section for "Neoplasms, hereditary". Here you will find various hereditary oncology malignancies. If of interest, please ask your rep or our support team (ampliseq-designs@thermofisher.com) for a list of inherited oncology genes by tumor type or site. Note, AmpliSeq On-Demand panels are intended and supported only for inherited disease applications using whole blood.

  12. I can't find my disease of interest in the DRA, can you help?

    We currently only have a search function to find your disease of interest. We do not currently support a back-fill of the hierarchy based on a disease selection. Contact our support team (ampliseq-designs@thermofisher.com) if there are disease research areas you believe should be added or if you need help finding a DRA.

Ion AmpliSeq On-Demand – IGV Viewer

  1. What is the IGV viewer?

    The Integrative Genomics Viewer (IGV) is a visualization tool for interactive exploration of genomic data created by the Broad Institute. Information can be found on their website.   The results shown for each gene are from the in-house wet-lab quality control testing (S5, 530 chip). The green track shows regions of the genes where amplicons were designed to target for amplification. The yellow track shows the resulting coverage obtained after sequencing. Users can zoom in to a target of interest and infer expected coverage.

  2. 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 validation 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.

  3. 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.

  4. What is the scale on the Y-axis?

    The Y-axis represents the experimental coverage, which has been normalized to 100.

  5. 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 on a gene and the IGV viewer will be updated automatically and centered on that gene. This version of the IGV viewer is not searchable by coordinate, variant or ID.

  6. 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 will be highlighted in the “Missed regions (if any)” track. It may 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 will then be able to visualize the lower number of reads.

  7. Why do some amplicons have very few reads in the “Expected coverage” track, versus others that have lots of reads?

    In order to achieve the most coverage (sensitivity), there is a sacrifice on specificity. So in some instances primers may either bind less tightly or bind off-target, thereby reducing the number of amplicon reads at the desired region.

Ion AmpliSeq On-Demand – Spike-in Panels (a.k.a. Companion Panels)

  1. What are Spike-in Panels?

    Spike-in panels are high concentration Made-to-Order panels that are used to expand the panel content to include genes not currently available in On-Demand inventory. Select the “Learn more” link in your design page for more information. Note that we cannot make any guarantees on the performance of Spike-In panels or On-Demand panels when used in conjunction with a Spike-In panel. Although the risk for primer interaction is low, due to the nature of the panels being designed and manufactured separately and combined by the user, we cannot assure performance. Note that Made-to-Order panels are not wet-lab tested by our in-house R&D. We will do our best help troubleshoot if there are any issues that we believe are due to the design or manufacture of our panels.

  2. What is the benefit of a Spike-in Panel?

    Since the number of genes available as On-Demand genes is limited, a Spike-in panel enables a user to sequence all the targets initially wanted in a single target amplification reaction. Note that a Spike-In panel is limited to < 123 amplicons per pool, or 246 amplicons total for a 2 pool On-Demand panel.

  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 the loss of the performance guarantee. 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 cannot be designed from the On-Demand panel design page and are not supported as the On-Demand panel performance may suffer due to dilution effects. From a performance standpoint, since Spike-in panels are manufactured as Made-to-Order panels and are not wet-lab tested like On-Demand panels, we cannot guarantee performance. Adding a Spike-in panel to an On-Demand panel will void the guarantee and should be done only if the user accepts this limitation. We suggest the user spike-in to a smaller number of On-Demand reactions and test on known samples before continuing to a larger number of samples.

  4. How are Spike-in Panels different from Ion AmpliSeq On-Demand Panels?

    Spike-in panels follow our Made-to-Order process. They are synthesized de novo at every order and are not wet-lab tested nor do they have any performance guarantee. Depending on the number of amplicons, Spike-In panels are offered in 750 reactions (<= 96 amplicons) or 3000 reactions (> 96 amplicons).

    On the other hand, On-Demand Panels have optimized designs, have been pre-manufactured, and wet-lab tested. They are available in small reaction number batches (8 or 32 for Chef and 24 or 96 for manual). On-Demand Panels also contain data that can be visualized in our IGV viewer available on the design page.

Ion AmpliSeq Designer HowTo

  1. What are the input files for Ion AmpliSeq Designer?

    To submit human or mouse genomic targets for assay design submission, users can input a BED file of genomic regions of interest, or a Gene List file based on HUGO gene symbols and aliases.

  2. Which coordinate system should I use in my BED formatted files?

    The BED format files in AmpliSeq use the convention known as “zero-based, half-open” (ZBHO) coordinates, both for input and for output files. In contrast dbSNP and COSMIC use “one-based, inclusive” (OBI) coordinates. Notice then that compared to dbSNP and COSMIC, AmpliSeq coordinates will have a start coordinate one less than that shown on the dbSNP and COSMIC databases.

    When comparing coordinates in BED files between AmpliSeq and data from the UCSC browser, please be aware that the UCSC Genome Browser uses both coordinate systems: OBI in the web interface and ZBHO in their database and data downloads.

  3. What is the current turn around time for a submitted design with respect to the target size or the number of targets?

    A design of 250kb or less should be returned in less than 48 hours of submission. For designs over 250kb or a large number of targets, you should expect a longer turn around time.

  4. Is there any way to get genomic coordinates automatically for the regions we want, instead of having to manually type them into a form?

    Yes, you can generate BED formatted files by utilizing the UCSC Genome Browser export feature in the Table Browser section. See Working With BED Files.

  5. Can we upload FASTA sequences?

    Not at this time. We are currently exploring different methods for uploading regions to the Ion AmpliSeq Designer.

  6. Can I use Galaxy instead of UCSC or IGV?

    Yes. Any tools can be used to help you generate files for submission, but it is important to make sure the correct version of the genome is being used (hg19 for human, mm10 for mouse).

  7. What browsers are supported with this application?

    We support Firefox, Google Chrome, Safari, and Internet Explorer 9 and above.

  8. What can I do to ensure that an entire exon is covered in my design?

    If coverage obtained from the initial design is less than 100%, you can try to extend the primer further out into the intron to capture the whole exon. Primer regions are not considered covered, so placing padding may ensure that we are able to get good quality sequence at the ends of exons, and to get some sequence read into the splice junction regions.

  9. Is it possible to use the Ion AmpliSeq design to actually screen a large number of SNPs (up to a 1000 or more) in a large number of individuals (up to a 1000 or more)?

    Yes, Ion AmpliSeq Designer allows you to do SNP genotyping by sequencing. Alternatively, you can also consider Taqman SNP Genotyping Assays for a large number of samples.

  10. Is it be possible to use the designer to detect differences between methylated/non-methylated DNA?

    The Ion AmpliSeq Designer does not design primers for methylation experiments.

  11. Can the designer be used for targeted whole genome sequencing?

    The Ion AmpliSeq is used for targeted resequencing. It cannot be used to sequence whole genomes.

  12. Exactly what is provided as output to the assay designs?

    When you click on the Download Results button of your Results ready project/version, the following output files are generated in a compressed folder.

    File Name Details
    #_coverage_summary.csv Gene-specific and region-specific coverage details
    #_coverage_details.csv This file provides details of coverage by exon for targets submitted by CDS or CDS+UTR (targets submitted as regions cannot be decomposed into exon-equivalents, so they are not listed in this file). If a request has no CDS (or CDS+UTR) targets, then there is no information for creating this coverage_details.csv file.
    #_Submitted.bed BED file with the genomic coordinates submitted to design
    #_Designed.bed BED file of coordinates of what the application designed to
    #_Missed.bed BED file of coordinates that were missed by the designer
    #_384WellPlateDataSheet.csv Amplicon ID, forward and reverse primer sequences. This file uses "OBI" coordinates (see FAQ number 2 in the "Designer HowTo" section for details).
    #_hotspot.bed This file contains all the hotspots that overlap with designed.bed (specific to the design) and our internal hotspot file called "Universal_hotspot.bed". This file is only available for AmpliSeq HD designs (IAH) and Oncomine tumor specific panels (OAD if customized).
    #_amplicon_insert_size_histogram.png This image file provides the user with a histogram view of the insert-size distribution of all the amplicons found in the panel. The insert being the region between the primers, which is amplified as reads, which in turn are used for sequencing of the targeted region.
    #_amplicon_size_histogram.png This image file provides the user with a histogram view of the amplicon size distribution of all the amplicons found in the panel. The amplicon being the targeted region including the primer information at the 5' end and 3' end.
    plan.json This file contains information to automatically configure a run plan for the panel, when the panel's files are directly downloaded from the Torrent Server 3.6

    It is possible to use the primers designed by AmpliSeq with a third party oligo synthesis provider. However, the primers manufactured by Thermo Fisher are optimized for their use with the Ion Torrent platform.

  13. When I submit a UCSC .bed file with exons from a few genes the user interface estimation of the size of my design is very large. Why? how can I prevent that?

    The user interface does not check for duplicate regions or any overlaps of the regions submitted in a .bed file. The UCSC .bed files typically contain duplicate regions for many quasi-identical transcripts. Too many overlapping regions may lead to a wrong estimate which may prevent the submission if the target size exceeds the currently allowed limit of 500 Kb.

    A simple and effective way that may help to prevent this, is by running the UCSC .bed file through the program mergeBed from the BEDTools suite. This will create equivalent regions in a smaller .bed file.

  14. Which is the largest design that I can submit to AmpliSeq?

    The largest design that can be submitted directly to the pipeline is at most 500 Kb. However the pipeline is capable of processing designs up to 5 Mb, but such designs are predictably costly and take up a large number of computational resources.

    In the cases of submissions larger than 500 Kb, the user will be contacted by email requiring more details about his/her interest in that particular, design and the design will be put on hold until the contact has been made.

  15. What is different in AmpliSeq.com 7.2?

    The DNA made-to-order pipeline in AmpliSeq 7.2 incorporates a number of algorithm improvements developed within the Custom Solutions Group at Thermo Fisher. These improvements have been validated on a large number of designs, and provide improved coverage and accuracy.

  16. Will the changes in AmpliSeq.com 7.2 always improve coverage?

    In the majority of cases, AmpliSeq.com will provide more coverage than previous versions. However, the new algorithm also implements additional checks and the quality of candidate oligos and amplicons, in particular, checking for specificity and insert uniqueness. In some less common scenarios, for example in cases of low-complexity regions of DNA, the algorithm may not be able to find amplicons meeting these quality checks to cover a region. In this situation, the reported coverage may be lower than previous versions, though actual amplicon performance should be improved.

  17. Will I still see multiple solutions for my design request?

    AmpliSeq.com 7.2 will continue to generate multiple solutions corresponding to different stringency levels and numbers of pools. However, we have found that generating solutions with different target amplicon lengths were not needed in most cases. Consequently, Ampliseq.com will now only generate solutions for the optimal amplicon length for the desired sample type. This only applies to AmpliSeq DNA made-to-order. See the next question for further details.

  18. Which designs will be impacted by the changes?

    In 7.2 the changes will affect AmpliSeq DNA made-to-order releases for human (hg19, GRChg38) and mouse (mm10) genomes. Improvements for additional genome support, including support for custom genomes, and support for AmpliSeq HD, are planned for a future release.

  19. If I already created a draft design, would I still get all the solutions for all the amplicon sizes as I did before?

    No. Once you're ready to submit your design for calculation, you will be prompted to select the desired amplicon size, and only solutions for the selected amplicon size will be generated.

  20. When using the Copy Amplicons functionality sometimes I get extra pools or errors. Why does this happen and what can I do to get past the error?

    When using the copy amplicons functionality, the system will attempt to create a solution which includes all the requested amplicons, if necessary adding additional pools, up to a maximum of 5 pools, in order to accommodate overlapping amplicons. If it is not possible to create a solution using the maximum number of pools then an error is raised. In this case the user must remove some overlapping amplicons/targets in order to create a valid solution.

  21. What is the maximum number of characters that I can use to describe a region name and which characters are not supported

    Region names can be at most 70 characters long, and may not contain any of the characters '[', ',', ';', '=', '"', ''', '\', '|', '/', ']' or space.

  22. Are there any restrictions that I should be aware of when uploading target bed files?

    Uploaded target bed files must have the extension '.bed' (lower case). There are no other restrictions.

  23. On occasion, there are gene symbols that are not recognized by the system, what can I do in this case?

    If a gene-symbol is not currently in the known list of aliases then it will be necessary to upload the specific regions included in the gene, in order to design a solution for the gene.

  24. For AmpliSeq Custom Made-to-Order, can I copy an amplicon from an FFPE design to a cfDNA design?

    Yes, please refer to the following compatibility table to understand the changes done during the Copy amplicons event:

    Source DNA Type Initial Draft DNA Type New Modified Draft DNA Type
    cfDNA (140bp) cfDNA (140bp) No modification
    cfDNA (140bp) FFPE (175bp) or Standard (275 or 375bp) No modification
    FFPE (175bp) cfDNA (140bp) FFPE (175bp)
    FFPE (175bp) FFPE (175bp) or Standard (275 or 375bp) No modification
    Standard (275bp) Standard (275bp or 375bp) No modification
    Standard (275bp) cfDNA (140bp) or FFPE (175bp) Standard (275bp)
    Standard (375bp) Standard (375bp) No modification
    Standard (375bp) cfDNA (140bp) or FFPE (175bp) or Standard (275bp) Standard (375bp)
  25. For AmpliSeq HD Custom Made-to-Order, can I copy an amplicon from an FFPE design to a cfDNA design?

    Yes, please refer to the following compatibility table to understand the changes done during the Copy amplicons event for AmpliSeq HD:

    Source DNA Type Initial Draft DNA Type New Modified Draft DNA Type
    cfDNA (125bp) cfDNA (125bp) No modification
    cfDNA (125bp) FFPE (175bp) No modification
    FFPE (175bp) cfDNA (125bp) FFPE (175bp)
    FFPE (175bp) FFPE (175bp) No modification
  26. Can I copy amplicons from an AmpliSeq Custom Made-to-Order design to an AmpliSeq HD Custom Made-to-Order draft design?

    Yes, please refer to the following compatibility table to understand the changes done during the Copy amplicons event where the draft design is for AmpliSeq HD:

    Source DNA Type Initial AmpliSeq HD Draft DNA Type New Modified Draft DNA Type
    cfDNA (140bp) cfDNA (125bp) FFPE (175bp)
    cfDNA (140bp) FFPE (175bp) No modification
    FFPE (175bp) cfDNA (125bp) FFPE (175bp)
    FFPE (175bp) FFPE (175bp) No modification
    Standard (275 or 375bp) cfDNA (125bp) Only amplicons <200bp will be copied, others will be ignored. Draft will be converted to FFPE type (175bp) to accommodate as many amplicons as possible.
    Standard (275 or 375bp) FFPE (175bp) Only amplicons <200bp will be copied, others will be ignored. Draft will remain as FFPE type (175bp).
  27. Can I copy amplicons from an AmpliSeq HD Custom Made-to-Order design to a regular AmpliSeq Custom Made-to-Order draft design?

    No, it is not possible to copy amplicons from an AmpliSeq HD design to a regular AmpliSeq design.

Ion AmpliSeq Designer Primer Design Bioinformatics

  1. How does the software accommodate intronic regions?

    When the user submits a gene to design, only exons are used as targets. If you wish to design across the whole gene (exons and introns) the user needs to 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 need to be 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 this might change in the future and a small overlap might be possible.

  4. For the 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 validated?

    Each primer pool goes through a rigorous process to meet strict design specifications. During the design of our pipeline, we validated a substantial number of our custom assays though wet lab testing.

  6. If I submit two continuous regions (175 - 225 bp range each) combined as one BED file, is it possible to get the designed primers for the overlapping region?

    If an overlapping region is submitted to the design pipeline, internally the region is concatenated and treated as a single region for design, thus there will be no overlap. The two regions are reported back in the UI as submitted. While it is possible that an amplicon might be prorated twice, once in each of the original regions, this amplicon (and its primers) only occurs once in the design (see the plate file).

  7. What is a superamplicon?

    A superamplicon is created when two forward PCRs joined to form one large amplicon. The pooler algorithm in the pipeline separates primers into separate pools to minimize this.

  8. The BED file specifications state that in a BED file the chrStart number is zero-indexed and the chrEnd number is not included in the feature. Are 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 0-99.

  9. Can you describe more about 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 that allows removal of any primer dimer formed along with the majority of the primer itself from the amplified template. This makes sequencing very efficient by not wasting bases on non-informative primer sequence and allows for very clean sequencing reactions.

  10. Do your designs take into account the presence of pseudogenes?

    Yes. The pipeline first attempts to design primers that only match the target, and not the pseudogene (or duplicate) version(s). If the target gene is not covered in the initial 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.

  11. If two amplicons overlap, do the primers produce a big product in addition to two small ones?

    The pooling step in the design is optimized in order to minimize the interference between overlapping amplicons. Hence, overlapping amplicons would be segregated into different pools.

  12. Why my gene is not accepted for design?

    There are several reasons that explain why this happens:

    1. A gene must be part of the UCSC Reference Gene dataset
    2. A gene must have at least one coding transcript
    3. A gene must not map to more than one genomic location (this includes pseudoautosomal genes (PAR1,2) )
    4. A gene must not map to un-assembled contigs or alternate assemblies - examples for human include: chrUn_gl000228, chr4_gl000194_random and chr6_cox_hap2 (see the UCSC FAQ on chrN_random tables )
  13. Which sequence versions does AmpliSeq use in its computations?

    DNA

    Human Genome* - Dec. 2013 (hg38, GRCh38.p2)

    Human Genome* - Feb. 2009 (hg19, GRCh37)

    Mouse Genome* - Dec. 2011 (mm10, GRCm38)

    Gene targets correspond to RefSeq v74

    Hotspots targets correspond to dbSNP v146 (for human and mouse) and COSMIC v86 (available only for human genome hg38)

    Hotspots targets correspond to dbSNP v138 (for human and mouse) and COSMIC v86 (available only for human genome hg19)

    RNA

    Human RNA Canonical RefSeq Transcripts* - Feb. 2009 (hg19, GRCh37)

    HGNC Database, HUGO Gene Nomenclature Committee (HGNC), EMBL Outstation - Hinxton, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SD, UK http://www.genenames.org, 11/2012

Human Genome version hg38

  1. Is hg38 the same genome version as GRCh38?

    Yes, they are the same version of the human genome. GRCh38 stands for “Genome Reference Consortium Human Reference 38” and it is the primary genome assembly in GenBank; hg38 is the ID used for GRCh38 in the context of the UCSC Genome Browser.

  2. Essentially, how is hg38 different from hg19?

    The hg19 build is a single representation of multiple genomes. The hg38 build provides alternate sequences (“alt_sequences”) for some genomic regions for which their variability prevents adequate representation by one single reference.

  3. How is the hg38 reference used by Thermo Fisher Scientific software different from the references publically available from places like UCSC or NCBI?

    • The version used by our software is based on GRCh38.p2 (http://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.28)

    • Unplaced, Alternate and Unlocalized contigs are listed as separate chromosomes and ordered first by chromosome localization, then by the alphabetic order of the Genbank accession of the contigs.

    • Repeat and SNP locations are soft-masked into lower case letters, while the ambiguous IUPAC bases, duplicated centromeric arrays, and chrY PAR regions are hard masked into 'N's.

    • It contains chr1-22, chrX, chrY, and chr22_KI270879v1_alt.

    • Contig chr22_KI270879v1_alt is hard masked except region 269814-279356 (1-based).

    • Gene GSTT1 is located at chr22_KI270879v1_alt:270308-278486.

  4. I know there are many more “alt_sequences”, why is that your version of hg38 only considers one of those?

    Our version of hg38 only considers the chr22_KI270879v1_alt. This alt chromosome contains gene GSTT1 that was part of chr22 in hg19. This was an internal decision which was made to enable standardization of the genome reference for use across multiple businesses within our organization.

  5. Can I download the hg38 files from NCBI and use them directly for my analyses of Ion sequencing data?

    We strongly recommend that you download our version of the hg38 from our website. This version is the one that is assumed in all of our software applications and has been tested for compatibility.

  6. Do you have a conversion tool from hg19 coordinates to hg38 coordinates?

    At this moment we do not offer any conversion tools. We recommend our users touch base with their own bioinformatics experts for further guidance.

  7. Can I analyze old assays or panels with the new hg38 build?

    No. The old assays and panels were created using hg19 as a reference and should be analyzed with the tools and analysis pipelines created for hg19.

  8. Can I still design, order and analyze old AmpliSeq panels based on hg19?

    Yes. The pipelines and tools for using hg19 as reference for design and analysis are still available.

  9. Can I still annotate my old variants (based on hg19) with Ion Reporter Software?

    Yes. The variant calling workflow based on hg19 will be available in Ion Reporter. If your design was created using hg38, then you can also call and annotate variants using Ion Reporter.

  10. Can I copy amplicons from an hg19 design to an hg38 design (or vice versa)?

    No. Amplicons from a custom design can only be copied to another custom design associated with the same reference. It is not possible to copy amplicons to a custom design associated with a different reference even if both references are human.

  11. Will there be a new version of the Ready-to-Use and Community AmpliSeq Research Panels based on hg38?

    Not at this moment. The off-the-shelf panels (ie exome), On-Demand panels and Community panels will still be based on hg19. Conversion of pre-designed panels may be considered in the future based on market demand.

  12. Will there be a version of the Oncomine Panels based on hg38?

    Not at this moment. The Oncomine panels will still be based on hg19.

  13. Are your Ion Reporter Software annotations based on hg38 or hg19?

    If your AmpliSeq design has been created using hg38 as a reference, then you can create an ad-hoc workflow in Ion Reporter for analysis. All analysis and annotations will take in consideration hg38 as a reference. However, at this moment there are no hg38 workflows in Ion Reporter. The tools for analysis and the annotations for hg19 will still be available.

RNA AmpliSeq

  1. Why does the Ion AmpliSeq RNA pipeline not accept the entered Gene Symbol?

    The pipeline recognizes HGNC approved gene symbols, previous gene symbols, and synonyms. It is case sensitive. Please check the Gene Symbol entered for typos and then search for the Gene Symbol on the HGNC website , to confirm that is a valid entry. The pipeline requires that the Gene Symbol entered is unambiguous, meaning that it resolves to a single HGNC approved gene symbol.

  2. Why does the Ion AmpliSeq RNA pipeline not produce designs to comprehensively target all RefSeq transcripts of the entered Gene Symbol?

    The pipeline uses strict criteria in order to design a single assay per gene that will result in consistent amplification while minimizing the risk of amplifying genomic DNA, this is done by targeting splice sites between exons. If there is not a splice site that is shared between all RefSeq transcripts of the entered Gene Symbol or if a passing assay could not be designed to the most common splice site then an assay will be produced that is compatible with a subset of the RefSeq transcripts for the entered Gene Symbol.

  3. Why does the Ion AmpliSeq RNA pipeline not accept the entered RefSeq accession?

    The pipeline attempts to resolve a single hg19 genomic alignment, where an exception is made for pseudoautosomal alignments, for each RefSeq accession. Unrecognized or Invalid RefSeq transcript accessions can result when the entered accession has been permanently suppressed or was not successfully resolved to a single hg19 genomic alignment.

  4. Why does the Ion AmpliSeq RNA pipeline produce a design that is not specific to the entered RefSeq transcript accession?

    The pipeline will design a single most-inclusive assay for the gene that corresponds to the entered RefSeq transcript accession. To achieve this, the pipeline will attempt to design an assay to the most common splice site found in the RefSeq accessions for the gene.

  5. Does the AmpliSeq RNA pipeline design assays for Mouse genes/transcripts? Fusion detection? Allele expression?

    No. These are among the list of features that are on the development roadmap and will be included in subsequent releases of the Ion AmpliSeq RNA pipeline.

  6. Is there a way in the AmpliSeq RNA pipeline to specify genomic regions to design against?

    No. The RNA pipeline does not allow specifying genomic regions for design.

  7. Can I input my own FASTA sequences to design against?

    No. The RNA pipeline does not allow the entry of FASTA sequences for design.

  8. Why the AmpliSeq RNA pipeline designed.BED file isn’t compatible with UCSC genome browser?

    The data on which AmpliSeq RNA pipeline is based on, is the NCBI's RefSeq sequences. A consequence of this is that all coordinates in the .BED files correspond to RefSeq coordinates that are not recognized by the UCSC genome browser. However the .BED files produced by the RNA pipeline are fully compatible with other pieces of IonTorrent software.

  9. Why is there a restriction on the minimum and the maximum number of amplicons in my design?

    The minimum of 12 amplicons is due to manufacturing process limitations. The exception to this limitation is AmpliSeq On-Demand which allows < 12 amplicons/pool in the spike-in design. Note, if an MTO DNA design panel has fewer than 48 amplicons it is subjected to our minimum order quantity policy of 48 amplicons (96 oligos) and its associated pricing.

    The maximum number of amplicons is based on validation test results that demonstrated good gene expression dynamic range on a PGM run. A good dynamic range means good sensitivity to detect extremes in the expression of genes in a given sample.

  10. How can I find out which is the TaqMan assay (if any) corresponding to my RNA targets?

    The last two columns of the IAD#_DataSheet.csv file (included in the download results files for an RNA design) reports the preferred TaqMan assay that can be used for verification of the particular target and classification of the TaqMan assay with 2 possible values:

    • Classification 1: The recommended TaqMan Gene Expression Assay targets the same exon or exon-exon boundary as the Ion RNA AmpliSeq Design.

    • Classification 2: The recommended TaqMan Gene Expression Assay targets the same set of RefSeq Accessions as the Ion RNA AmpliSeq Design.

    Follow this link for instructions on how to get the corresponding TaqMan assay.

  11. AmpliSeq RNA warns me about including high expressed genes in my design. How do you know they are high expressers?

    The high expressed genes were selected from rank-ordered lists of whole transcriptome RNA-Seq expression data derived from universal human reference RNA (UHRR, Stratagene). UHRR is comprised of purified RNAs from 10 distinct human cell lines and has been shown to be an accurate and reproducible standard for comparison of gene expression data. This reference RNA has been utilized by the highly referenced Microarray Quality Control (MAQC) consortium as well as the more recent Sequence Quality Control (SEQC) study. There is familiarity among microarrays users primarily as well as some RNA-Seq customers around the MAQC samples which also bolsters the decision to use this RNA for the rank ordered list.

    Data Collection: Whole transcriptome sequence data was collected from both PGM and Proton runs using Ion 318 and Ion P1 chips, respectively.
    Since UHRR represents several different tissues and we have a wealth of internal RNA-Seq data from this sample, we found UHRR to be a reasonable sample type for determining a common list of highly expressed genes.

    References

    UHRR product information

    MAQC Study Webpage (FDA)

    MAQC Paper

Ion AmpliSeq Exome Panel

  1. Is the exome panel suitable for somatic samples?

    While the depths of coverage on a 550 chip may be suitable for somatic variant calling with the Ion AmpliSeq exome panel, the recommendation for the 550 chip will be for germline variant calling only. Due to the 125-275 bp amplicon designs, we cannot guarantee the performance of the panel on degraded DNA from samples such as cfDNA or FFPE.

  2. Are there future plans for a new Ion AmpliSeq exome kit to be compatible with FFPE samples?

    We are not currently considering this option, but its implementation will depend on the demand for it. Please contact your rep or our support team for product or feature requests.

  3. How many reactions are supported by the Ion AmpliSeq Exome bundle kits?

    Each Ion AmpliSeq Exome bundle (PN A38262 and A38264) has enough oligos for 8 reactions, or 8 exomes. are each provided with a 24 reaction Ion AmpliSeq Library kit Plus (PN 4488990) - which supports 8 exomes.

  4. What is the shelf life of the bundle kit?

    PN A38262 and A38264 have a shelf life of 15 months due to the library kit. The exome oligo plates are stable for 3 years. Please contact customer service for shelf life questions or COA documents.

Ion Panels

  1. Does having "hotspots" in my panel mean that only those variants can be detected?

    No. The hotspots are variants that we have tested can be detected using the panels and the Torrent Variant Caller for producing the call (the _hotspots.bed file forces the TVC to make a call whether or not the variant is present in those particular hotspots).

Performance

  1. How do we measure on-target bases (specificity)?

    On-target bases is the percentage of total sequenced bases that mapped to target regions. This metric reflects the percentage of bases from the amplicons that were designed, synthesized, and pooled that also generated sequence data that mapped back to the target regions.

  2. How is coverage of all targets ensured, in terms of both target submission and wet chemistry (assay conversion)?

    The Ion AmpliSeq Designer takes into account many different parameters to compute the best set of amplicons to cover a target region. The ability to maximize in silico coverage depends upon factors such as repetitive regions and sequence complexity. The Ion AmpliSeq 2.0 User Guide provides guidance on how many amplicons can be combined to either the Ion 314 chip, the Ion 316 chip, or the Ion 318 chip. Our coverage uniform is >85% of amplicons is 0.2X of the mean coverage. If the mean (or average) coverage is 2000X, then 85% of the amplicons have a depth of coverage that is > 400X.

  3. Experimentally how does one manipulate coverage? If I want 100 X, then later I want 500X, what experimentally is manipulated to achieve this?

    If you want additional coverage from your experiment, you could always run a larger chip or multiple chips. For example, you could simply take the same library that was constructed in your initial experiment, and then run another template prep and sequencing run on a subsequent chip in your second experiment. Currently, the recommendation to achieve ~500X coverage is to run ~1kb on an Ion 314 Chip, 50kb on an Ion 316 Chip, and 500kb on an Ion 318 Chip.

Ion AmpliSeq – Oligo Ordering

  1. How many reactions do I receive when I order my DNA AmpliSeq design in Tubes Only format?

    Refer to the link for a table describing the number of reactions that you can expect, depending on the size of your panel.

  2. How many reactions do I receive when I order my DNA AmpliSeq design in Tubes and 384-well plate format?

    Refer to the link for a table describing the number of reactions that you can expect, depending on the size of your panel.

  3. How many reactions do I receive when I order my RNA AmpliSeq design in Tubes Only format?

    Refer to the link for a table describing the number of reactions that you can expect, depending on the size of your panel.

  4. Are RNA AmpliSeq designs available in Tubes and 384-well plate format, like DNA designs?

    No, not currently. The only format available for RNA designs is in Tubes Only format.

  5. Why do we provide pooled and plated primers?

    To maximize convenience and flexibility. Pooled primers can be used immediately. Plated oligos can be used to: 1) Rebuild the same pool, 2) Rebuild a pool with fewer primers.

  6. Can I add a few more genes to a set of previously ordered primer?

    Currently, the only way to do this is to duplicate the target list in a new version, add the new genes and resubmit. As long as the same design attributes are set (CDS/all exons 150/200) as previously used, the genes from the previous set will have the same design.

  7. I am not in the United States. Will my order be shipped directly to my lab, or first to a local Thermo Fisher distribution center, then to my lab?

    This depends. With the exception of orders from Europe that are processed in the U.K., all other international orders are first processed by the North America customer service team, who sends the form to the local customer service team for verification and final changes. The local customer service team works with the customer to determine the best route for a shipment, and the decision is made by the local customer service team. Shipment to a distribution center is slower, but it is significantly less expensive for Thermo Fisher, and could potentially result in fewer customs issues and tax charges. Direct shipment is generally faster, but this adds additional shipping costs for Thermo Fisher, and there may be customs and/or tax implications.

  8. If I have a few regular primers for a region and I know they are working, can I add these primers to my AmpliSeq design?

    Yes, you will need to use the "Copy Amplicons" function to ensure the amplicons used in the original design are used for your new design.

  9. Can I add primers manually, afterwards, to completely cover a region?

    Yes, you can add amplicons to your assay using the process known as "Spike-In". Check with your local Field Support team for guidance on how to add the new targets, and make sure you make the selection for 50X primer concentration at the time of ordering.

  10. Is there a minimum order for AmpliSeq?

    Ion AmpliSeq Custom Panels range from 12 amplicons* to 6,144 amplicons per tube. The minimum of 12 amplicons is due to manufacturing process limitations. The exception to this limitation is AmpliSeq On-Demand which allows < 12 amplicons/pool in the spike-in design. Note, if an MTO DNA design panel has fewer than 48 amplicons it is subjected to our minimum order quantity policy of 48 amplicons (96 oligos) and its associated pricing.

    Target regions can be as small as 1 kb and can go up to 5 Mb.

  11. Are custom primer pairs sent only in 384-well plates, or are they also available all premixed in one tube?

    Both. Each custom primer pool is delivered as both a pre-pooled tube and as individual primer pairs plated into 384-well plates. Small orders of up to 96 amplicons per pool will contain 750 pre-pooled reactions and individual primer pairs sufficient for 1,500 reactions. Larger orders of more than 96 amplicons per pool will contain 3,000 pre-pooled reactions and individual primer pairs sufficient for 6,000 reactions.

  12. How can I find out the status of the design submission for my Ion AmpliSeq Custom order?

    Email us at genomicorders@thermofisher.com or call 1-800-955-6288, x46636. Please use your Ion AmpliSeq Design ID number when referring to your order. Please contact your local customer service outside of North America.

  13. What is the Custom oligo cancellation policy?

    There is no guarantee of cancellation of a custom oligo order. Please contact your local customer service representative for more information and options. On occasion, customer service can intercept an order and is able to cancel it prior to synthesis. You must call ASAP: 1-800-955-6288, x46636.

Troubleshooting and Validating

  1. If one of the important genes missed in the coverage or was off the target, do we need to start all over? Could we just redesign that gene and plex it again?

    No, you do not need to start all over. You can add, delete and edit genes or regions from the design results. You can even create a new version of the same design, with iterative modifications, and continue to resubmit the designs.

  2. Is the recommendation to validate the sequencing data with Taqman SNP? (Direct sequencing vs indirect TaqMan assay?)

    Variant confirmation can be done with other platforms, including Taqman SNP Genotyping Assays and Sanger sequencing–capillary sequencing.

  3. Suppose we are targeting a region and the AmpliSeq Designer suggest a design consisting of 2 primer pools. For each sample, should we prepare a library for each amplification (each pool) or should we combine the 2 amplifications (the products of the 2 amplified pools), and then do the library?

    If your design results in multiple pools, each pool should be treated independently when making libraries. You can pool the libraries together prior to the template preparation step using Ion OneTouch Systems. So if you have 2 primer pools for your panel, and plan to test 1 of the DNA samples using a single chip (assuming that your target size and required sequencing coverage can be met with a single chip), then you will have 6 libraries, three of those libraries use one barcode and the remaining 3 libraries use a second barcode.

  4. Could the tumor-amplified DNA and normal-amplified DNA be loaded onto the same chip, then the sequences be separated out during analysis?

    Yes, you can pool your tumor and normal samples together into a single chip run (assuming that your target size and required coverage can be achieved in a single chip). Many people perform differential pooling so that the coverage of the normal sample is lower than the tumor sample.

  5. We see a large variation in the coverage of different regions of our panel. What could you recommend?

    We recommend using the Ion AmpliSeq Library Plus Kit as it is expected to produce higher library yield, increased uniformity and result in more robust library amplification than the standard Ion AmpliSeq 2.0 Library Kit.

  6. Is there any way to determine if a variation is true or is a mistake introduced by the polymerase?

    There are a number of ways to perform orthogonal validation of mutations found by NGS, including TaqMan Genotyping Assays in standard or digital PCR formats, TaqMan Mutation Detection Assays for specific somatic mutations, or Sanger Sequencing using Capillary Electrophoresis.

  7. Do you have plans to make your instrument and related products for in vitro diagnostic use?

    Yes, the PGM Dx is Class II, 510k cleared and the Oncomine Dx Target Test assay is Class III approved assay in the US.

  8. How many bp are the primers separated from the target region, for example, by an exon?

    To ensure that an entire exon is covered, by default we add 5 bp of padding up and down-stream of the selected target region to allow room to place the primers. Padding ensures that we are able to get good quality sequence at the ends of the exons and to get some sequence read into the splice junction regions. Primer regions are not considered covered. Therefore, if coverage obtained from the initial design is less than 100%, we can try once more to extend the primer further out into the intron to capture the whole exon.

  9. Could you target multiple pathogens, while filtering out off-target human genome amplification?

    Designs for pathogens are not currently supported for Ion AmpliSeq Designer.

  10. Could I do two or three different amplifications and then pool before going into library prep?

    It is possible to run 3 different AmpliSeq designs each with barcodes and combine them going into Template Prep.

  11. Can I import pre-design PCR primer sets and validate if they work?

    Target regions from pre-designed PCR primer sets can certainly be imported and submitted for design into the Ion AmpliSeq Designer. The specific parameters of Ion AmpliSeq Designer may result in primer sets that are different from initially pre-designed PCR primer sets but are optimized for use with the Ion AmpliSeq Technology.

  12. Why am I getting fewer targets in my results than what I submitted in my .BED file?

    Due to quality control considerations after submission, amongst other properties, the .BED file is reviewed for duplicates. Once the duplicates have been removed and other filters have been passed, the (probably reduced) file is accepted into the pipeline.

Access code

  1. What is an Access Code and what is it used for?

    An access code is used as part of the gateway for communication between AmpliSeq Designer, and the Ion Torrent analysis software, which can be either Torrent Suite or Ion Reporter.

  2. What can I do if I lose my Access Code?

    If the user loses their Access Code, the recommendation is to generate a new one.

  3. Can I create my own Access Code?

    Yes, as long as the Access Code fulfills the minimum requirements, a custom Access Code is accepted.