Non-targeted fusion detection method
In the non-targeted fusion detection method, the panel primers are used to detect fusions between novel combinations of known driver and partner genes. The sequencing reads are mapped to a broader reference, such as the whole‑exome. Mapping the reads to a broader reference allows for the detection of multiple configurations of driver and partner genes as well as detection of novel breakpoints between the known partner and driver genes.
Low confidence non-targeted fusions are identified in VCF output files with key value pairs as follows.
Type of non-targeted fusion identified |
Definition |
Key value pair |
|---|---|---|
Out-of-frame sequence |
The expected non-targeted sequence from the amplicon reference file is used to check whether the sequence is in frame. If the non-targeted sequence is out of frame (compared to the original partner or driver genes), the key-value pair NT_FUSION_IN_FRAME=FALSE is included in the VCF file. |
NT_FUSION_IN_FRAME[1] Values |
Partner-partner fusion |
Non-targeted fusions are identified if one of the genes in an isoform is not a driver gene. The input reference BED file includes annotations for driver genes, such as, in the case of EML4-ALK.E6A19.COSF1296.2, the DRIVER_GENE=ALK. By default, the software uses a 3p-partner of a targeted fusion isoform as the driver gene if that targeted isoform BED entry does not include the DRIVER_GENE parameter, as is the case with earlier panel files and also a few targets in later panel files. In this case, the software will check if at least one of the two genes involved in non-targeted fusion is a 3p gene. |
NT_FUSION_DRIVER_INVOLVED[2]. Values |
Secondary isoform fusion |
In some samples, a non-targeted fusion isoform might be detected in a sample while additional targeted isoforms with the same gene pair are also detected (that is, isoforms that are defined in the panel). In this case, the non-targeted fusion isoform should be identified as NT_FUSION_SECONDARY=TRUE, if its read count coverage is below <x% of the reads of any of the other isoforms with the same gene pair. By default, the threshold for secondary isoforms is <5% of another targeted fusion isoform. |
NT_FUSION_SECONDARY[3] Values |
Additional attributes that indicate whether the non-targeted fusion is out of frame, involves partner-partner fusion, or is a secondary isoform, are added to the VCF files. This information is used by the Oncomine™ Variant Annotator plugin to annotate variants in Ion Reporter™ Software for each Oncomine™ assay and the Ion AmpliSeq™ HD Library Kit.