Tumor Mutational Burden

You can compute Tumor Mutational Burden (TMB) if running the SNV caller in Tumor-Only or Tumor-Normal Mode.

DRAGEN uses the following steps to compute TMB:

1. Generates variant calls with DRAGEN tumor-normal mode.
2. Identifies eligible regions. Only regions that meet the specified minimum required coverage are eligible.

You can specify custom regions of interest using the command line.

3. Filters the following variants.
• FAIL variants
• Mitochondrial variants
• MNVs
• Variants that do not meet the minimum depth (DP) threshold. Use the --vc-callability-tumor-thresh command line option to specify the threshold value.
• Variants that do not meet the minimum variant allele threshold. Use the --tmb-vaf-threshold command line option to specify the threshold value.
• Variants that fall outside the eligible regions.
• Germline variants. Variants are determined as germline if they meet one of the following criterion.
• Variants in the sample VCF with VAF > 0.9.
• Variants with a population allele count ≥ 10 that are observed in either the 1000 Genome or gnomAD database.
• If more than five surrounding variants are germline.
• Tumor driver mutations. Variants with a population allele count ≥ 50 are treated as tumor driver mutations. You can specify the cosmic driver threshold using the tmb-cosmic-count-threshold command line option.
4. Identifies nonsynonymous variants. Only nonsynonymous events are included. Nonsynonymous consequences include:
• feature_elongation, feature_truncation, frameshift_variant
• incomplete_terminal_codon_variant, inframe_deletion, inframe_insertion
• missense_variant, protein_altering_variant, splice_acceptor_variant
• splice_donor_variant, start_lost, stop_gained, stop_lost, transcript_truncation
5. Computes TMB and nonsynonymous TMB using the following equations.
• TMB= Filtered Variants/ Eligible Region
• Nonsynonymous TMB=Filtered Nonsynonymous Variants / Eligible Region

Command-line Option

Description

--enable-tmb true

Enables TMB. If set, the small variant caller, Illumina Annotation Engine, and the related callability report are enabled.

--qc-coverage-region-1

Specify the coding regions to use.

--vc-callability-tumor-thresh

Specify the minimum coverage for coding regions. Coding regions that do not meet the threshold value will not be used for the final TMB calculation.

--qc-coverage-tag-1

Set --qc-coverage-tag-1 to tmb to execute DRAGEN TMB.

--qc-coverage-reports-1

Set --qc-coverage-reports-1 to callability to execute DRAGEN TMB.

--enable-variant-annotation=true

--variant-annotation-assembly

--variant-annotation-data

Enables Illumina Annotation Engine. For more information on selecting the correct assembly and downloading reference files, see Illumina Annotation Engine.

[Optional]--tmb-vaf-threshold

Specify the minimum VAF threshold for a variant. Variants that do not meet the threshold are filtered out. The default value is 0.05.

[Optional]--tmb-db-threshold

Specify the minimum allele count (total number of observations) for an allele in gnomAD or 1000 Genome to be considered a germline variant. Variant calls that have the same positions and allele are ignored from the TMB calculation. The default value is 10.

The TMB values are output to <output prefix>.tmb.metrics.csv. The file format uses the following CSV column convention, similar to other metric CSV files.

Metric

Definition

Eligible Region (Mbp)

The specified custom regions that meet the minimum coverage threshold.

Filtered Variant Count

Remaining variants after VAF, variant type, and germline filters.

Filtered Nonsyn Variant Count

Filtered variants that are filtered further to include only nonsynonymous events.

TMB

Filtered variants by the eligible regions. ?

Nonsyn TMB

Filtered nonsynonymous variants by the eligible regions.