Exon tile fusion baselines

When an exon tile fusion baseline is applied to an analysis workflow, Ion Reporter™ Software can model the amplicon expression variation within a gene. The exon tile fusion baseline is used to normalize a test sample, correct the coverage in exon-tile amplicons, and identify the presence or absence of expression imbalance in a gene.

An exon tile fusion baseline is a TXT file that contains:

the normalized expression values of exon-tile amplicons for genes with imbalance assays
the median normalized expression for each amplicon

Ion Reporter™ Software includes predefined exon tile fusion baselines. You can also create an exon tile fusion baseline workflow preset in the software. You can later add the exon tile fusion baseline to an analysis workflow and use it as a baseline control for analyses.

An exon tile fusion baseline must include at least 48 fusion-negative RNA samples. Various parameters and thresholds used in the baseline computation determine the inclusion or exclusion of samples in the baseline for each of the genes, for example, ALK, RET, NTRK1, that are assayed in exon-tiling analysis workflows. The primary criteria for inclusion in the baseline is sufficient coverage across the exon-tile amplicons of a given gene. Samples with low coverage across the exon-tiling amplicons for one of the genes are excluded from the baseline.

Note: A sample can qualify for inclusion in a baseline of one gene but can be excluded from the baseline of another gene. For example, if a gene is not expressed in the tissue of the sample, the sample is excluded for that gene.

A summary table of the samples that are included and excluded in the exon tile fusion baseline for each gene is contained in a log file called createBaseline.ReadCounts.summary. For more information about file downloads, see Variants file downloads. Here is an example exon tile fusion baseline summary file.

Genes that are included in exon-tiling imbalance analysis workflows. Each row summarizes the imbalance baseline composition status of a gene.
Each sample is represented by a column. In this example, sample 24 is included in the baseline for ALK, FGFR2, and HMBS, but excluded for NTRK1, NTRK2, NTRK3, and RET.
The last column, with the header total, lists the total number of valid baseline samples per gene.
When a baseline results in fewer than 5 valid samples for one or more genes, a warning message is shown. In this example, the warning message applies to ALK.

Create an exon tile fusion baseline workflow preset

You must use at least 48 fusion-negative RNA samples to create an exon tile fusion baseline workflow preset.

In the Workflows tab, click Presets, then click Create PresetExon Tile Fusion Baseline.
In the Baseline Type step of the Create Exon Tile Fusion Baseline workflow bar, select the Fusion Panel to use for the exon tile fusion baseline.
Note:
- The baseline type is set to Exon Tile Fusion Baseline and cannot be changed.
- The reference genome is set to hg19 and cannot be changed.
Click Next.
In the Parameters step, do not change parameters from the default settings unless you understand how the change can affect the analysis. Click Next.
In the Samples step, select the samples to be used in the baseline creation.

You must select at least 48 or more fusion-negative RNA samples. If you do not see samples of interest in the table, see Sample definition for information about how to upload or define a sample.
Click Next.
In the Confirm step, enter a name, or accept the default name, then enter an optional description for the baseline.
Click Launch.

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