Frequently Asked Questions


Select an FAQ Topic to Explore:

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 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 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 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 275bp, 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@lifetech.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@lifetech.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@lifetech.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@lifetech.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. How can multiple labs share a single assay design (one design, multiple synthesis)?

    You can use the Sharing feature to share a read-only URL link to designs. To access this feature, click on the More Actions + button at the top right of the “ My Designs” tab when displaying a design with "Ready" status.

  5. 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.

  6. Can we upload FASTA sequences?

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

  7. 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).

  8. What browsers are supported with this application?

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

  9. How is the amplicon design criteria determined?

    Currently, Ion AmpliSeq Designer allows users to choose between 150 bp and 200 bp amplicon size for each design. You can also use the More Solutions + button to toggle between 200bp/150bp amplicon size designs. The amplicon size includes the primer sequences and the insert regions. The option for 150 bp is what we recommend for FFPE DNA and 200 bp for normal DNA. The Ion PGM System can be used to sequence 1x100 bp and 1x 200 bp.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Exactly what is provided as output to the assay designs? Can those files be used with a third party oligo synthesis provider?

    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
    IAD# _coverage_summary.csv Gene-specific and region-specific coverage details
    IAD# _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.
    IAD# _Submitted.bed BED file with the genomic coordinates submitted to design

    IAD#_
    Designed.bed
    BED file of coordinates of what the application designed to
    IAD# _Missed.bed BED file of coordinates that were missed by the designer
    IAD# _Missed_Detail.bed BED file containing the reasons of the missing coverage
    IAD# _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).
    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.

  15. 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.

  16. 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.

  17. The size of my panel that is reported in the UI changed after the AmpliSeq release of March 31st, 2014 (AmpliSeq 3.4), Why? Did my design actually changed? Is it safe to order this panel?

    The changes in the reported size are due to a change in the algorithm used by the UI to estimate the size and nothing else. The change was necessary as the older algorithm was underestimating the panel size for several instances. Nothing actually happened to your design, its actual size did not change. If available, you can compare the design files downloaded time ago when the design was generated, to the files available for download now: they must be identical. The manufacture of AmpliSeq panels is actually based on those files produced by the designer and so a panel ordered now should be identical to a panel previously ordered.

  18. 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.

  19. 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.

  20. 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.

  21. 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.

  22. 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.

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. Can I use a subset of the Fixed Panels for a custom design?

    Yes. Clicking on the Customize Panel button for the Panel design you are working with will create a starting template where you can delete or add genes or regions.

  7. For 200 bp designs, should the BED file submitted be within a 175 - 225 bp range?

    No, you do not have to select the gene coordinates (BED file) to be 175 - 225 bp. The Ion AmpliSeq Designer designs the primer pairs and provides the appropriate BED file and primer sequences to be approximately 200 bp amplicons.

  8. 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).

  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 because the fraction of the genome covered is so low compared to the hg19 reference that we are not showing enough decimal places?

    If a full reference was used, and you have only tens of Mbs, the coverage is so low that we don't track that far out.

  10. 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.

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

  12. 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.

  13. 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.

  14. 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.

  15. 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 )
  16. 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 v63

    Hotspots targets correspond to dbSNP v146 (for human and mouse) and COSMIC v76 (available only for human genome hg38)

    Hotspots targets correspond to dbSNP v138 (for human and mouse) and COSMIC v68 (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

    * These files are available for download at the AmpliSeq Ion Community website

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. What criteria were used to select the hotspots in the Ion cancer panels?

    Given the high number of mutations in the genes in the panels, filtering was applied in order to include as "hotspots", only those COSMIC mutations (COSMIC v60) observed in more than 1% of the tested samples or in 2 or more samples. For the Colon & Lung panel, some Non-COSMIC mutations considered relevant were included as well.

  2. 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 (download the guide in the Ion Community Developer Access area). 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.

Oligo Ordering

  1. 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.

  2. 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.

  3. 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.

  4. 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?

    No, not at this time.

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

    No, not at this time. We use specially modified primers, so standard primers will not allow for library construction.

  6. 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.

  7. 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.

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

    Email us at genomicorders@lifetech.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.

  9. 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. Can I start with the AmpliSeq Cancer Panel and make my own version with a few extra custom amplicons?

    You are not able to take an existing primer pool and combine with a new primer pool because they would have been developed independently. However, you can create a panel using gene symbols or gene regions from an existing Ion AmpliSeq Ready-to-use Panel, and add and delete some genes, and then submit for design. The BED files and gene lists for ready-to-use panels are available at Ion Community.

  7. 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.

  8. 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.

  9. 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.

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

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

  11. 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.

  12. 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.

  13. 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.