Copy and edit an analysis workflow template

To analyze Ion AmpliSeq™ HD sequencing data in Ion Reporter™ Software, you need to create a user-defined analysis workflow for use with Ion AmpliSeq™ HD panels. To create a user-defined analysis workflow, first copy a predefined Ion AmpliSeq™ HD analysis workflow template, then add a target regions file, and any available hotspots file or CNV baseline. For RNA samples, you need to also add fusion panel files to the user-defined analysis workflow. This procedure must be used when creating an analysis workflow for custom Ion AmpliSeq™ HD research panels.

After you create the user-defined analysis workflow, you can select the analysis workflow in Torrent Suite™ Software to transfer data automatically to the appropriate Ion Reporter™ Server and use the analysis workflow for the data analysis in Ion Reporter™ Software.

  1. In the Workflows tab, click Overview.

    For information about available Ion AmpliSeq™ HD analysis workflow templates, see instead Analysis workflow templates for Ion AmpliSeq HD.

  2. In the Workflows table, select the Ion AmpliSeq™ HD analysis workflow template that you want to copy, then click  ActionsCopy.

    The Edit workflow bar opens to the Research Application step. Review the Research Application and Sample Group.

    When you copy analysis workflow templates, you cannot change the Research Application or Sample Group.

  3. Click Next, then in the Reference step, select or upload the appropriate files.

    Option

    Description

    Select a file that was previously uploaded to Ion Reporter™ Software.

    Select the Target Regions and, if needed, Hotspot Regions files from the dropdown lists.

    Upload the entire package of the Ion AmpliSeq™ HD panel files directly from AmpliSeq.com.

    When you use this option, a target regions file and any available hotspot regions or fusion files specific for the panel are uploaded and available in the Workflow Presets screen.

    Click AmpliSeq Import.

    1. In the Import for AmpliSeq dialog box, select the Custom Panel tab for Ion AmpliSeq™ or Ion AmpliSeq™ HD Made-to-Order, or Ion AmpliSeq™ On Demand panel files, or select the Fixed Panel tab for Community or Ready-to-Use panel files.

    2. Enter an account user name.

    3. In the Password field, enter the access code. For more information, see 3.c.

    4. Click List My AmpliSeq Panels.

    5. From the list, select the fusion panel file that you want to import.

    6. Click Import.

    Import a target regions file that you previously downloaded from AmpliSeq.com and saved to the local storage.

    1. Under the Target Regions list, click Upload.

    2. Click Select file, browse to, then select the target regions BED file, then click Open.

    3. Select Ion AmpliSeq™ HD, then click Upload.

    (Optional) Upload a hotspot regions file that was previously uploaded from AmpliSeq.com and saved to the local storage.

    1. Under the Hotspots Regions list, click Upload.

    2. Click Select file, browse to, then select the hotspot regions BED file, then click Open.

    3. Select Ion AmpliSeq™ HD, then click Upload.

    If you use a panel that detects fusions, upload a ZIP file that was previously downloaded from AmpliSeq.com and saved to the local storage.

    1. Under the Fusion Panel list, click Upload.

    2. Click Select file, browse to, then select the fusions ZIP file, then click Open.

    3. Select Ion AmpliSeq™ HD, then click Upload.

  4. Click Next.
  5. In the Annotation step, ensure or select an annotation set and, if applicable, select a MyVariants database, then click Next.
  6. In the Filters step, select a filter chain from the Filter Chains list to change the default filter chain for analyses that use this analysis workflow. If you do not change the filter chain, the default filter chain is saved for the new analysis workflow.

    Option

    Description

    Called Variants and Controls

    This filter chain reports all variants (either hotspots or novel) that pass the filter and are not called as reference or NOCALL. Variant types include SNV, INDEL, MNV, CNV, LONGDEL, FUSION, EXPR_CONTROL, ASSAYS_5P_3P, RNA_HOTSPOT, GENE_EXPRESSION, RNAExonVariant, ProcControl, FLT3-ITD, and RNA Exon Tiles.

    Called Hotspot Variants and Controls

    This filter chain reports all hotspot variants that pass the filter and are not called as reference or NOCALL. Variant types include SNV, INDEL, MNV, and RNA Exon Tiles.

    Variant Matrix Summary

    Select this filter chain for analysis results that contain the same set of variants that are included in the Variant Matrix Analysis visualization for Ion AmpliSeq™ HD analysis results.

    The Variant Matrix Summary filter chain filters in:

    • Positive hotspot calls

    • Positive gene-level CNV GAIN or LOSS calls

    • FLT3-ITD variants

    • Non-wild-type intragenic structural variations

    • For TagSeq Breast and TagSeq Pan-Cancer assays: non-hotspot variants in TP53 with QUAL >50 and Allele Frequency > 1.0 (Breast) or 0.5 (Pan Cancer)

    This filter chain allows analysis results to be exported. You can then import the results into another software application for further analysis.

    Filter variant types include: SNV/INDEL, CNV, fusions, and RNAExonVariants.

    If you use the Oncomine™ Myeloid MRD analysis workflow, a FLT3-ITD variant subtype named Combined can be reported in analysis results. The Combined subtype is a value for the sum of all reported FLT3-ITD variant allele frequencies.

    Oncomine™ Variants

    Select this filter chain to show only the variants that are annotated with the Oncomine™ Variant Annotator plugin. For more information, see Oncomine Variant Annotator plugin criteria.

  7. Click Next.
  8. In the Copy Number step, select a copy number baseline from the Baseline list, if applicable, then click Next.
  9. In the Plugins step, select a plugin, then click Next.

    In the Plugins step, the Oncomine™ Variant Annotator plugin is selected by default. This plugin adds annotations for variants that are relevant to cancer with Oncomine™ Gene Class and Oncomine™ Variant Class information. This plugin is included by default with the Ion AmpliSeq™ HD analysis workflow templates. If you import the VCF file of analysis results into Oncomine™ Reporter, these annotations are included in a report that is generated from that software.

  10. In the Final Report step, select or confirm the final report template that is selected in the list, then click Next.
  11. In the Parameters step, make any required changes, then click Next.
  12. In the Confirm step, name the analysis workflow, enter an optional description, then click Save Workflow.

The newly created analysis workflow is added to the list of analysis workflows in the Workflows tab in the Overview screen.