Fusions parameters

You can adjust the following fusions parameters to optimize analysis results when you create or edit Ion Reporter™ Software analysis workflows.

IMPORTANT! Use the default parameter settings unless you are an advanced user.


Parameter	Description
Main tab
Sensitivity	Sensitivity. Allowed values: Fixed values, only one of can be applied. High–the algorithm requires 60% overlap between reads and reference sequence with at-least 50% exact matches in the overlap. Medium– the algorithm requires 70% overlap between reads and reference sequence with at-least 66.66% exact matches in the overlap. Low–the algorithm requires 80% overlap between reads and reference sequence with at-least 75% exact matches in the overlap.
Minimum Read Counts for Fusions	Threshold on the minimum number of valid reads aligned to specific fusion isoform sequence to call the isoform as present, if the normalized read count is also greater than the threshold. Example : If count of a target is >20, the target is called present. Allowed values: ≥0 Integers only
Minimum Normalized Read Counts for Fusions	Threshold on minimum normalized read counts threshold required to call a fusion isoform as present. Allowed values: ≥0 Float values
Minimum Total Valid mapped reads	Minimum number of total valid mapped reads required to qualify a sample as valid and to proceed with the analysis. Allowed values: ≥0 integers only
Make calls based on Imbalance Score	If this flag is set to true, Imbalance scores are used to make fusion presence, absence, or nocall calls. Allowed values: True or False (boolean)
Minimum number of Valid pools	For multipool RNA pools, specify the minimum number of pools in a sample that have to pass QC to qualify that sample as valid and proceed with the analysis. Example: If a panel has two pools, use value = 2 to specify that both pools needs to have sufficient number of reads to qualify that sample. Similarly use value = 1 to proceed with the analysis even if one pool fails. Allowed values: ≥1 integers only
Minimum Total Valid mapped reads Per Pool	Minimum number of total valid mapped reads in each pool (if there are multipool RNA panels) to qualify that primer pool as valid. Allowed values: ≥0 integers only
Advanced tab
Minimum Read Counts for Non-Targeted Fusions	Threshold on minimum number of valid reads aligned to a nontargeted fusion sequence to call the fusion as present. Example : If the count of a non-targeted isoform is >250, it is reported as present-nontargeted. Allowed values: ≥0 integers only
Minimum Read Counts for Controls	Threshold on minimum number of valid reads aligned to specific expression control sequence required to call it as present. Example : If the read count of an expression control is >15, it is called present. Allowed values: ≥0 integers only
Minimum Total Control reads	Minimum number of housekeeping control reads required to calculate Imbalance scores for 5p3p assays. Allowed values: ≥0 integers only
Maximum Imbalance for Negatives	If the Imbalance score of any driver gene is less than this value, the sample is called fusion negative for that gene. Allowed values: Text field. String value in specific format as shown in the default value. This verifies the input of a Regular expression.
Minimum Imbalance for Positives	If the imbalance score of any driver gene is greater than this value, the sample is called fusion positive for that gene. However, there is a grey zone between maximum and minimum values where scores are called nocall. If they are equal, there is no grey zone. Allowed values: Text field. String value in a specific format as shown in the default value. This verifies the input of a Regular expression.
Minimum Isoform Counts for Imbalance	If the sum of counts from all the isoforms of that driver gene is greater than this number, thresholds set by Maximum Imbalance for Negatives with evidence from Isoforms and Minimum Imbalance for Positives with evidence from Isoforms are used for the imbalance scores. Allowed values: Text field. String value in a specific format as shown in the default value. This verifies the input a Regular expression.
Maximum Imbalance for Negatives with evidence from Isoforms	If the imbalance score of any driver gene is less than this value, the sample is called fusion negative for that gene. Allowed values: Text field. String value in a specific format as shown in the default value. This verifies the input of a Regular expression.
Minimum Imbalance for Positives with evidence from Isoforms	If the imbalance score of any driver gene is greater than this value, the sample is called fusion positive for that gene. However, there is a grey zone between maximum and minimum values where scores are called nocall. If they are equal, there is no grey zone. Allowed values: Text field. String value in a specific format as shown in the default value. This verifies the input of a Regular expression.
Estimate max crosstalk	Maximum percentage of spill-over reads that could be seen in any sample due to reasons like barcode crosstalk. Allowed values: ≥0 (% values) float values
Analysis configuration file	A tab-separated file specific to each panel that enables users to set individual target specific thresholds for the following properties, as applicable for that type: Minimum read count Minimum normalized read count Minimum wild type ratio Make calls Do not report Max read count negative Allowed values: Path to a tab-separated file
Keep Intermediate files	Turn this flag on to keep the intermediate files generated when using the fusions analysis. Allowed values: True or False
Report non-targeted fusions	If this flagged is turned off, any nontargeted fusions detected are not reported in the output VCF file. Allowed values: True or False
Minimum Read Counts for Gene Expression targets	Threshold on minimum number of valid reads aligned to specific gene expression target to call it as present. Allowed values: ≥0 integers only
Minimum mean read length for valid SampleQC	If the average read length computed from all the reads in the sample is less than the value specified, that sample is not qualified to be valid and results are not reported. This is an additional SampleQC metric. Other QC metrics are minimum total valid mapped reads and minimum number of valid pools. For example, a recommended value is 50 bp. Allowed values: Integers only
Use pool Specific normalization	For multipool RNA panels, use this flag to specify whether read counts are to be normalized to total reads in each pool separately or to total reads in the sample. This also applies to calculation of wild type ratio and normalized count in gene metrics for RNAExonVariant targets. Allowed values: True or False
Minimum Molecular Family Consensus Size	Minimum number of reads with same tag required to form a functional family. Suggested value between 3 and 7. Impact: Increasing values make variant calls less sensitive but more specific. Allowed values: Integers only
Minimum Molecular Family Count	Minimum number of variant supporting functional families required to make a call. Impact: Increasing values make calls less sensitive but more specific. Allowed values: Integers only
Minimum Family Coverage per Strand	Minimum required coverage of reads on each strand in a bidirectional molecular tag family. Allowed values: Integers only
Minimum Number Of PC Amplicons Required To Pass QC	Minimum number of process (or expression) control amplicons containing equal or more families than fusions.min.fam.count required to pass quality control. Allowed values: Integers only
Minimum Number Of PC Amplicons Required To Fail QC	Maximum number of process (or expression) control amplicons containing equal or more families than fusions.min.fam.count required to fail quality control. Allowed values: Integers only
Minimum read counts for RNAExonVariants	Minimum number read counts for RNAExonVariant targets to be called as present. This value is used only in cases where present/absent calls are made for RNAExonVariant targets. Allowed values: Integers only
Minimum Molecular Family Count for RNAExonVariant	Minimum number of variant supporting functional families required to make a call for RNAExonVariant targets. This value is used only in the cases where present/absent calls are made for RNAExonVariant targets. Allowed values: Integers only
Minimum Imbalance Score for the RNAExon Tile Assays	Minimum imbalance score for calling 'imbalance positive' from RNA exon tiling assays in a driver gene. Positive calls also depend on the P value for imbalance. Allowed values: ≥0 float values Note: Gene-specific parameters are enabled in the properties.txt file.^[1]
Minimum p-value for Imbalance Score for the RNAExon Tile Assays	Maximum statistical significance P value for calling 'imbalance positive' from RNA exon tiling in a driver gene. Positive calls also depend on imbalance scores. Allowed values: 0-1 float values Note: Gene-specific parameters are enabled in the properties.txt file that is included in downloaded analysis files.^[1]
Minimum average read counts for all the Exon Tiling assays in a Driver Gene	Mean coverage of a driver gene with RNA exon tiling assays. Measured per gene, as the total valid mapped reads counts from all exon-tiling assays divided by the number of exon-tiling assays Allowed values: ≥0 float values Note: Gene-specific parameters are enabled in the properties.txt file.^[1]
Number of weak amplicons	The RNA Exon Tiling amplicons beyond the breakpoint are required to have sufficient expression. This parameter defines an upper boundary to allow a defined number of exon tiling amplicons that fail to reach a coverage threshold. (Coverage that is below than 20x coverage the maximal amplicon in the gene).^[1]
Minimum number of exon tiling amplicons flanking predicted breakpoints	A parameter that restricts the position of the breakpoint by the minimum number of flanking exon tiling amplicons. Samples in which breakpoints are predicted at very close proximity to the 3’ and 5’ – without sufficient number of exon tiling amplicons on both sides – will result in No Calls.^[1]
Minimum number of Wild type assays detected for RNAExonVariants	Minimum number of wild type assays detected for RNAExonVariants. This parameter is for use with Ion AmpliSeq HD and TagSeq analysis results and analysis workflows only.^[1] Allowed values: ≥0 integers only
Use either family counts only or read counts only to make calls	Use this flag to make calls based on either family counts (molecular counts) or read counts. Turn this flag on to enable dynamic calls using either metric, with molecular counts analyzed first, followed by read counts. Turn this flag off to require both metrics to make calls. This parameter is for use with Fusion, RNAExonVariant, and ProcessControl fusion types. For more information, see Data types for gene fusions analyses. Allowed values: True or False

¹ For more information, see Analysis configuration file for gene fusion analysis.