Annotation parameters

You can modify the following annotation 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

Analysis options

Functional Annotations For All Alleles

Flag to include functional annotations for genotype-positive alleles only (false) or all reported alleles (true) for variants.

Allowed values: True or False

Suggested trial value: False

Use IUPAC Single Letter Code for Amino Acid

Use True for IUPAC single letter code for amino acid. Use False for three letter code.

Allowed values: True or False

Suggested trial value: False

Annotation Statistics and Reporting General Options

dbSNP Hit Level

Flag to control specificity of dbSNP annotations. 'overlap' matches all annotations whose loci overlap with variant. 'locus' matches all annotations whose loci start at variant locus. 'allele' matches all annotations that are 'locus' matches plus have at least one allele in common with variant. 'auto' hit level matches the most specific hit level possible to the annotation which could be any of the overlap, locus, allele or genotype hit levels.

Allowed values:

  • overlap

  • locus

  • allele

  • auto

Suggested trial value: overlap

ClinVar Hit Level

Flag to control specificity of ClinVar annotations. 'Overlap' matches all annotations whose loci overlap with variant. 'Locus' matches all annotations whose loci start at variant locus. 'Allele' matches all annotations that are 'locus' matches plus have at least one allele in common with variant. 'Auto' hit level matches the most specific hit level possible to the annotation which could be any of the overlap, locus, allele or genotype hit levels.

Allowed values:

  • overlap

  • locus

  • allele

  • auto

Suggested trial value: allele

COSMIC Hit Level

Flag to control specificity of COSMIC annotations. 'Overlap' matches all annotations whose loci overlap with variant. 'Locus' matches all annotations whose loci start at variant locus. 'Allele' matches all annotations that are 'locus' matches plus have at least one allele in common with variant. 'Auto' hit level matches the most specific hit level possible to the annotation which could be any of the overlap, locus, allele or genotype hit levels.

Allowed values:

  • overlap

  • locus

  • allele

  • auto

Suggested trial value: locus

VariantDB Hit Level

Flag to control specificity of VARIANTDB annotations. 'Overlap' matches all annotations whose loci overlap with variant. 'Locus' matches all annotations whose loci start at variant locus. 'Allele' matches all annotations that are 'locus' matches plus have at least one allele in common with variant. 'Genotype' matches all annotations that are 'allele' matches where the genotypes also match. 'Auto' hit level matches the most specific hit level possible to the annotation which could be any of the overlap, locus, allele or genotype hit levels.

Allowed values:

  • overlap

  • locus

  • allele

  • auto

Suggested trial value: locus

ExAC Hit Level

Flag to control specificity of ExAC annotations. 'Overlap' matches all annotations whose loci overlap with variant. 'Locus' matches all annotations whose loci start at variant locus. 'Allele' matches all annotations that are 'locus' matches plus have at least one allele in common with variant. 'Auto' hit level matches the most specific hit level possible to the annotation which could be any of the overlap, locus, allele or genotype hit levels.

Allowed values:

  • overlap

  • locus

  • allele

  • auto

Suggested trial value: overlap

Gene Extension Size

Gene extension is the number of bases upstream and downstream of a transcript's start and end positions that should include the regulatory and control regions.

Allowed values: 0 to unlimited

Suggested trial value: 1,000

Splice Site Size

The 5' splice site of an exon is the small intronic region immediately upstream, which depends on the strand. Its size in bases is given by splice site size.

Allowed values: 0 to unlimited

Suggested trial value: 2

Note: Prior to Ion Reporter™ Software 5.10, we defined splicesite_5 and splicesite_3 as exon-centric. This was contrary to the common convention of splice site nomenclature that was intron-centric and would exchange the splicesite_5 and splicesite_3 designations. In Ion Reporter™ Software 5.10, splicesite_5 and splicesite_3 refers to the intron locations. In earlier releases of the software, you might have seen a 3' splice site in your results; you will now see a 5' splice site and vice versa.

Tumor Mutational Burden tab

Tumor Mutational Burden Filter Chain

Filters out the potential germline variants and retain the somatic variants for tumor mutational burden calculation after the variant calling and variant annotation is completed for the analysis.

To enable tumor mutational burden calculations on DNA samples, you must also edit, or copy and edit, either: any DNA–Single Sample, or DNA and Fusions–Single Sample analysis workflow. For more information, see Enable tumor mutational burden calculation in existing analysis workflows.

To enable the tumor mutational burden calculation for any other analysis workflow, you must select one of the available filter-chain options.

Allowed values:

  • TMB (Non-germline Mutations). If you use an analysis workflow for a Tumor Mutation Load Assay, TMB (Non-germline Mutations) is selected by default.

  • Mutation Load (Somatic Mutations)

  • Additional custom filter chains (if created on the server)

Suggested trial value: TMB (Non-germline Mutations)

Minimum Base Coverage

The minimum depth of base coverage required for a variant to be counted for TMB calculation.

Allowed values: 0 to unlimited

Suggested trial value: 60

TMB Variant Minimum Allele Frequency

The minimum alternate allele frequency for a variant to be included for TMB calculation.

Allowed values: 0 to 1

Suggested trial value: 0.05

You can use this parameter to reduce the affect of deamination in low-quality FFPEs and achieve a higher minimum allele frequency for a tumor mutational burden calculation. For more information, see Reduce the impact of deamination in low-quality FFPEs.

TMB Variant Region Type

The type of region to include for TMB calculation.

Allowed values:

  • Exonic regions only

  • Total target regions (exonic + intronic regions)

Suggested trial value: Exonic regions only

TMB Variant Type

The variant types to be included for TMB calculation.

Allowed values:

  • SNV

  • INDEL

  • MNV

Suggested trial value: SNV and INDEL

TMB Variant Effect Type

The variant effect types to be included for TMB calculation.

Allowed values:

  • missense

  • frameshiftDeletion

  • frameshiftInsertion

  • nonframeshiftDeletion

  • nonframeshiftInsertion

  • nnsense

  • stoploss

  • synonymous

  • unknown

Suggested trial value: Start with the default selections.

Deamination QC Threshold

The deamination value above which a sample is deemed failed for TMB reporting.

Allowed values: 0 to unlimited

Suggested trial value: 100

Calibration Minimum Cutoff

The lower limit of the range at which the germline calibration is applied.

Allowed values: 0 to unlimited

Suggested trial value: 25

Calibration Maximum Cutoff

The upper limit of the range at which the germline calibration is applied.

Allowed values: 0 to unlimited

Suggested trial value: 100

TMB Germline-Filter Calibraition Factor: Slope

The user-supplied value for the slope of the linear curve to which the number of somatic variants will be calibrated.

Allowed values: 0 to unlimited

Suggested trial value: 1.4637

TMB Germline-Filter Calibraition Factor: Intercept

The user-supplied value for the intercept of the linear curve to which the number of somatic variants will be calibrated.

Allowed values: −2,000 to 2,000

Suggested trial value: 0

TMB-Low Threshold

The TMB (mut/mb) threshold below which a sample is defined as TMB-Low.

Allowed values: 0 to unlimited

Suggested trial value: 10

TMB-High Threshold

The TMB (mut/mb) threshold above which a sample is defined as TMB-High.

Note: A TMB score that is above the TMB-Low Threshold and below the TMB-High Threshold is defined as Intermediate.

Allowed values: 0 to unlimited

Suggested trial value: 20

TMB Standardization

Apply the standardization of the observed TMB value to fit a linear curve.

Allowed values: On or Off

Suggested trial value: Off

TMB Standarization Factor: Slope

The user-supplied value for the slope of the linear curve to which the observed final TMB value will be adjusted.

Allowed values: 0 to unlimited

Suggested trial value: 0

TMB Standarization Factor: Intercept

The user-supplied value for the intercept of the linear curve to which the observed final TMB value will be adjusted.

Allowed values: -2,000 to 2,000

Suggested trial value: 0