Fusions parameters

You can adjust the following Fusions parameters to optimize your 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.

Suggested trial value: Medium

Minimum Read Counts for Fusions

Threshold on the minimum number of valid reads aligned to specific fusion isoform sequence in order to call the isoform as present, provided that 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

Suggested trial value: 20

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

Suggested trial value: 0

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

Suggested trial value: 20,000

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)

Suggested trial value: True

Minimum number of Valid pools

For multipool RNA pools, specify the minimum number of pools in a sample that have to pass QC in order 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 in order to qualify that sample.

Similarly use value = 1 to proceed with the analysis even if one of the pools failed.

Allowed values: ≥1 integers only

Suggested trial value: 2

Minimum Total Valid mapped reads Per Pool

Minimum number of total valid mapped reads in each pool (in the case of multipool RNA panels) in order to qualify that primer pool as valid.

Allowed values: ≥0 integers only

Suggested trial value: 0

Advanced tab

Minimum Read Counts for Non-Targeted Fusions

Threshold on minimum number of valid reads aligned to a nontargeted fusion sequence in order 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

Suggested trial value: 250

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 a expression control is >15, it is called present.

Allowed values: ≥0 integers only

Suggested trial value: 15

Minimum Total Control reads

Minimum number of housekeeping control reads required to compute Imbalance scores for 5p3p assays.

Allowed values: ≥0 integers only

Suggested trial value: 1,200

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 user's input using a Regular expression.

Suggested trial value: ALK=0.001;RET=0.03;ROS1=0.2

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 user's input using a Regular expression.

Suggested trial value: ALK=0.015;RET=0.55;ROS1=0.5

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 user's input using a Regular expression.

Suggested trial value: ALK=5;RET=5;ROS1=5

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 user's input using a Regular expression.

Suggested trial value: ALK=0.001;RET=0.3;ROS1=0.15

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 user's input using a Regular expression.

Suggested trial value: ALK=0.01;RET=0.25;ROS1=0.5

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

Suggested trial value: 0.5

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

Suggested trial value:

Keep Intermediate files

Turn this flag on to keep the intermediate files generated when using the fusions analysis.

Allowed values: True or False

Suggested trial value: 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

Suggested trial value: True

Minimum Read Counts for Gene Expression targets

Threshold on minimum number of valid reads aligned to specific gene expression target in order to call it as present.

Allowed values: ≥0 integers only

Suggested trial value: 10

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

Suggested trial value: 0

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 within gene metrics for RNAExonVariant targets.

Allowed values: True or False

Suggested trial value: True

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

Suggested trial value: 2

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

Suggested trial value: default 2, suggested value between 2 and 10

Minimum Family Coverage per Strand

Minimum required coverage of reads on each strand in a bidirectional molecular tag family.

Allowed values: Integers only

Suggested trial value: 1

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

Suggested trial value: 2

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

Suggested trial value: 1

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

Suggested trial value: 20

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

Suggested trial value: 2

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

Suggested trial value: 2.0

Note: Gene-specific parameters are enabled in the properties.txt file. For more information, see Analysis configuration file for gene fusion analysis.

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

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

Suggested trial value: 30.0.

Note: Gene-specific parameters are enabled in the properties.txt file. For more information, see Analysis configuration file for gene fusion analysis.

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

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

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

Allowed values: ≥0 integers only

Analysis configuration file for gene fusion analysis