Sequence variant baselines

The filters that are used in Ion Reporter™ Software remove most sequencing errors. Although variant calling filters in the software suppress sequencing errors, it is possible that some errors can pass through the filters and be reported as false positives. However, in somatic panels, sequencing errors that survive filtering are reported as false positives that require investigation.

To decrease the number of potential false positives, you can create a sequence variant baseline for panels that detect somatic variants.

A sequence variant baseline is BED file that contains a list of alleles that are known sequencing errors. The baseline file records detailed properties for each sequencing error. The errors and properties are based on known germline normal samples. When added to a workflow, the baseline detects and filters out the errors in sequencing that can lead to false positive variant calls.

Ion Reporter™ Software includes predefined sequence variant baselines for use with Oncomine™ panels and other ready-to-use panels. You can also create a user-defined sequence variant baseline, which is especially useful for custom Ion AmpliSeq™ panels that do not include a predefined sequence variant baseline.

Create a Sequence Variant Baseline workflow preset

You can create a custom Sequence Variant Baseline workflow preset in Ion Reporter™ Software to help reduce noise. You can later add the custom baseline to an analysis workflow that you can use as a baseline control for analyses.

If you import the custom sequence variant baseline, the target regions file that was used to create the baseline must be available in the software to ensure that the imported baseline appears in the list of available sequence variant baselines. When you add the baseline to an analysis workflow, the same target regions files that is used to create the baseline must also be used in the analysis workflow.

The sequencing run or runs that are used to create the sample data must use the same Ion Torrent chip type and same Ion AmpliSeq™ panel. For example, if an SVB will be applied to a sequencing run that used a Ion 540™ and custom Ion AmpliSeq™ panel, then the samples that were used to create the SVB must have been sequenced with the Ion 540™ and same custom Ion AmpliSeq™ panel.

  1. In the Workflows tab, click Presets, then click Create PresetSequence Variant Baseline.
  2. In the Baseline Type step of the Create Sequence Variant Baseline workflow bar, select the baseline type that corresponds to the type of libraries that you use.

    Baseline type

    Description

    AmpliSeq Somatic

    Ion AmpliSeq™

  3. Select a reference genome.

    • GRCH38

    • hg19

  4. Select the Target Regions file that corresponds to the panel. If the target regions file is not available in the panel, you can upload the file.
  5. Click Next.
  6. In the Parameters step, do not change parameters from the default settings unless you understand how the change can affect your analysis. Click Next.
  7. In the Samples step, do the following:
    1. Select the samples to be used in the baseline creation.

      You need to add at least 40 samples, up to a maximum of 100 samples, to create the custom sequence variant baseline. All samples must be normal (non-tumor). Samples can be of either gender. The software examines gender data for each sample.

      If you do not see the samples in the table, see Sample definition for information about how to upload or define a sample.

    2. Select the Chip Type that was used for the sample data.

      All samples that you select for the sequence variant baseline must use the same chip type.

  8. Click Next.
  9. In the Confirm step, enter a name, or accept the default name, then enter an optional description for the baseline.
  10. Click Launch.

The baseline creation job is started and the baseline with its status now appears on the screen. When the job completes, the baseline is added to the Workflow Presets screen and is available to add to an analysis workflow. For more information, see Apply a sequence variant baseline workflow preset to an analysis workflow.