3'/5' Imbalance scores for assay calls

Panels that include 3'/5' amplicons generate 3'5 imbalance values. The imbalance values report the difference in expression between the 5' assay and the 3' assay of each driver gene. Samples that do not contain a fusion are expected to have similar expression of the 5' assay compared to the 3' assay of the driver gene. Samples that contain a fusion are often expected to have elevated expression of the 3' assay compared to the 5' assay.

The 3'/5' Imbalance values provide a measurement of the strength of fusion calls that involve ALK, RET, or ROS1 driver genes, whether included or not included in the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel.

The 3'/5' Imbalance score is therefore included for two alternate purposes:

• To confirm presence of a fusion from the ALK, RET, or ROS1 driver genes included in the panel, as shown in this figure:

  

• If none of the fusions targeted by the panel is detected, to provide evidence of a fusion other than those targeted by the panel but still including ALK, RET, or ROS1 driver genes.

The following approaches interpret the 3'/5' Imbalance values for each gene and are reliable only under the conditions that are described in the following sections:

• Read number impact on calculation sensitivity

• Sensitivity

• High 5' expression

Most samples that are tested for the 3'/5' Imbalance assay have been lung tumor tissue. Threshold settings for other sample types can be sensitive to varying expression of fusion gene and expression control genes.