Create a Variability Correction Informatics Baseline (VCIB) workflow preset

You can create a Variability Correction Informatics Baseline (VCIB) workflow preset in Ion Reporter™ Software to determine copy number changes in the sample of interest, without the use of a matched control. You can later add the custom copy number baseline to an analysis workflow that you can use as a baseline control for analyses.

You must use a minimum of 48 samples, and at least 6 of the samples must be normal, to create a Variability Correction Informatics Baseline (VCIB) workflow preset.

If you import a custom Variability Correction Informatics Baseline (VCIB), the target regions file that was used to create the copy number baseline must be available in the software to ensure that the imported copy baseline appears in the list of available copy number baselines.

  1. In the Workflows tab, click Presets, then click Create PresetCopy Number Baseline.
  2. In the Baseline Type step, make the following selections.
    1. Select the Baseline Type that corresponds to the type of libraries that you use. For more information, see Variability Correction Informatics Baseline (VCIB) baseline workflow presets.

      Baseline Type

      Description

      AmpliSeq HD

      Create VCIB baselines for Ion AmpliSeq™ HD libraries.

      AmpliSeq

      Create VCIB baselines for Ion AmpliSeq™ and Oncomine™ libraries (not Exome).

      AmpliSeq-Exome

      Create VCIB baselines for Ion AmpliSeq™ Exome libraries.

      TagSeq

      Create VCIB baselines for Ion TagSeq libraries.

      TargetSeq-Exome

      Create VCIB baselines for Ion TargetSeq™ Exome or other targeted libraries.

    2. Select a reference genome.
      GRCH38
      hg19
    3. Select the Target Regions file that corresponds to the panel.

      • If you have already uploaded a germline Target Regions <panelID>_Designed.bed file for the custom Ion AmpliSeq™ panel, select it from the dropdown list.

      • If the target regions file is not available in the dropdown list, click (Upload), then select the <panelID>_Designed.bed file of the panel that you imported from Ion AmpliSeq™ Designer to upload the file from your computer.

    4. If the Target Regions is not imported from Ion AmpliSeq™ Designer Use the following options to import AmpliSeq libraries and panel files.

      1. Click AmpliSeq Import.

      2. 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.

      3. Enter an account user name.

      4. In the Password field, enter the access code. For more information, see Generate an access code for AmpliSeq.com.

      5. Click List My AmpliSeq Panels.

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

      7. Click Import.

  3. Click Next.
    The CNV Detection Type step of the Create Copy Number Baseline workflow bar opens. Ensure that the Somatic (CNV VCIB) algorithm is selected for use with VCIB workflow presets.
  4. In the CNV Detection Type step, make the following selections to make a new baseline, or start from an existing baseline.
    • To create a new baseline, deselect the Start with an existing CNV baseline checkbox.
    • To use an existing baseline and augment the baseline with more samples, select Start with an existing CNV baseline. The target region file selected in the Baseline Type step is shown in the dropdown list. For more information, see Augment an existing VCIB baseline workflow preset.
  5. Click Configure parameters to configure the parameters if needed, then click Done. For more information about these parameters, see the user guide for the panel that you use, or contact your Field Bioinformatics Specialist.
  6. Click Next.
  7. In the Samples step, select the 48 samples to be used in the CNV baseline creation.
  8. Click Next.
  9. In the Confirm step, enter a name, or accept the default name, and enter an optional description for the baseline.
  10. Click Create Baseline.
When the baseline creation is complete, it is locked and shown in the Presets screen with a State of Successful and is available for use in the software. The new baseline can be added to an analysis workflow. For more information, see Create a new analysis workflowCopy and edit an existing analysis workflow, and Apply a copy number baseline workflow preset to an analysis workflow.

If the State of a copy number baseline is Failed, place the pointer over the row of the copy number baseline and click View Log to open the log in a separate browser tab, or click Download Log to download the log. The log file can help you troubleshoot the failure.

Use gender to call gains from expected copy number changes

You can use the following approach to call gains from expected copy number changes with analyses that are performed on Oncomine™, Ion AmpliSeq™, or Ion AmpliSeq™ HD cancer research panels that use analysis workflows that include the Variability Correction Informatics Baseline (VCIB).

A copy number baseline has a gender, either male or female. A sample also has a gender: male, female, or unknown (unknown is interpreted as female). Expected genomic copy number regions for these genders are defined in the ploidy files that are supplied by Ion Reporter™ Software within analysis workflows.

For example, create a copy number baseline with "male" normal human samples and chromosome X non-Pseudo Autosomal Regions (non-PAR) amplicons in the panel. The baseline copy number for the non-PAR chromosome X amplicons is expected to be 1. If a male sample is run using this baseline the relative copy number to the baseline should be 1:1 and is expected (normal) and should be reported accordingly. A female sample using this baseline should have a relative copy number of 2:1 as is expected (normal) and should be reported accordingly. Using a ploidy file, each amplicon is assigned to the baseline expected copy number found in the baseline ploidy file (male), in this example non-PAR chromosome X amplicons would have a copy number of 1. Running a sample with a female ploidy file, the non-PAR chromosome X amplicons has a copy number of 2, so the final copy number relative to the baseline is adjusted appropriately.

More precisely, if for amplicon *, expected baseline copy number = B, and expected sample copy number = S, after variability corrections, set l2r = log2 ratio of sample observation to the baseline.

Final CN = 2**(lrr) * B

For purposes of reporting at the CNV_ID level, S and B are used to determine how different Final CN is from expected for that sample. The actual algorithm is proprietary.

  1. Create a normal male and chrX PAR CNV baseline.

    For more information, see Create a Variability Correction Informatics Baseline (VCIB) workflow preset.

  2. Add the CNV baseline to an Oncomine™, Ion AmpliSeq™, or Ion AmpliSeq™ HD analysis workflow.

    For more information, see Apply a copy number baseline workflow preset to an analysis workflow.

  3. Launch the analysis.

    For more information, see Launch an analysis.

  4. Visualize the analysis.

    For more information, see IRGV & Generate Report tab or Visualize analysis results with Ion Reporter Software.

Copy number gains are reported on the Analysis Results screen and the Analysis Visualization screen, Variant Matrix tab. For more information about the results, see Reasons for NOCALL in a gene-level CNV.