Fusion calls in analysis results

Fusion calls describe a translocation of genetic material. The Genes (Exons) column reports the donor gene and the partner gene. The exon number for each gene is reported in parentheses. For the donor gene (the first one in the pair), exon number specifies the exon before the fusion. For the partner gene (the second one in the pair), exon number specifies the exon after the fusion.

For example, in the second row, the Genes (Exons) column reports EML4(6) - ALK(20):

• EML4(6) refers to exon number 6 on the EML4 gene (the donor gene in this example). Exon number 6 in the donor gene indicates that the fusion starts after exon 6.

• ALK(20) refers to exon number 20 in the ALK gene (the partner gene in this example). Exon number 20 in the partner gene indicates that the fusion starts before exon 20.

The Detection column reports Present for calls that are supported by read evidence. The default values for the thresholds can be changed and the threshold for read evidence support varies by call type.

• Fusions: Greater than 20 supporting reads are required to report Present.

• Expression controls: Greater than 15 supporting reads are required to report Present.

For example, if the threshold of the Detection column is set to greater than 20 reads for fusions and 250 reads for non-targeted fusions, then the following occurs:

• Absent: Either no evidence for the fusion is found or fewer than 21 reads support the fusion.

• Present: Greater than 20 reads provide evidence for the fusion.

• Present-Non-Targeted: Greater than 250 reads provide evidence for a fusion that is not explicitly included in the Ion AmpliSeq™ panel. A non-targeted fusion is a fusion for which the panel did not contain a specific primer pair, but it was picked up by a combination of two of the primers used for two different targeted isoforms. The fusion was not among the targeted isoforms and was not explicitly targeted by the panel. If there are a small number of reads providing evidence for such fusions (for example, less than a few hundred reads), these may not be important. If there is very strong evidence for non-targeted fusions, they may be worth following up with more detailed investigation. A literature search or a search of COSMIC or other databases may be helpful in determining whether such fusions have ever previously been observed, and may provide guidance in interpretation.

• No Call: Not enough evidence to determine if a fusion is present or not.

For TagSeq and Ion AmpliSeq™ HD analysis workflows: To make PRESENT/ABSENT calls, minimum molecular count threshold (default value : >=3) is applied for each target along with the minimum read count threshold (default value > =21). For these analysis workflows, the reason for the call, whether by molecular counts, read counts, or both, is listed in the Detection column in the Fusions table:

In Ion Reporter™ Software 5.16 and later, fusion calling in analysis workflows for Ion AmpliSeq™ HD and TagSeq panels and assays can be done through the use of either molecular (family) counts or read counts, and is based on the thresholds for each type of count for a particular fusion. When this parameter is set to True in the analysis workflow, the software calculates molecular counts and makes a Present call if the threshold value is met. If the threshold is not met, the software then calculates read counts to determine if the Present call can be made. Optionally, you can choose to require the threshold value of both molecular count and read count metrics to be met to make a Present call, which is the case when the parameter is set to False. The parameter which controls the way a call is made is named “Use either family counts only or read counts only to make calls”. The type or types of counts used to make the call is displayed in the Detection column of the Analysis Results screen. The impact of this parameter, when turned on, is the potential for increased fusion call sensitivity. This parameter has no impact on NoCalls.

Other information that is reported for fusions includes the following:

• The Locus column reports the start and end positions of the fusion transcript.

• The Read Counts column reports the number of reads that provide evidence for the fusion call. For ASSAYS_5P_3P, read counts are displayed in the order 5', 3'.

• The COSMIC/NCBI column provides links at which the fusion is described in these public web sites.

• The variant ID column reports our shorthand identifier for the fusion variant (or assay or expression control). Each target in the Ion AmpliSeq™ panel has a unique variant ID.

  An identifier contains information that is available in other fields in the results table.

  Information	Description, example
  Partner gene name	ALK
  Gene and exon number	A13, for exon number 13 in the ALK gene
  COSMIC ID, NCBI GenBank ID	COSF1198, AB374361
  Non-Targeted	Appended to the Variant ID for fusion calls that are not targeted by the Ion AmpliSeq™ panel. Example: SLC34A2-ROS1.S4R35.Non-Targeted

  Examples of Variant IDs for fusion calls (these examples are from demo data):

  • EML4-ALK.E6bA20.AB374362:

    • EML4: Donor gene

    • ALK: Partner gene

    • E6: Exon number 6 in the ELM gene ("E" for "ELM")

    • b: The second fusion (ordered by locus) with the same value in the Genes (Exons) column

    • A20: Exon number 20 in the ALK gene ("A" for "ALK")

    • AB374362: GenBank ID

  • SLC34A2-ROS1.S4R35.: Non-Targeted

    • SLC34A2: Donor gene

    • ROS1: Partner gene

    • S4: Exon number 4 in the SLC34A2 gene ("S" for "SLC34A2")

    • R35: Exon number 35 in the ROS1 gene ("R" for "ROS1")

    • Non-Targeted: Not included in the Ion AmpliSeq™ panel