Tumor Mutational Burden

DRAGEN supports Tumor Mutational Burden (TMB) in Tumor-Only or Tumor-Normal Mode.

It is important to note that in T/O mode germline variants must be identified and filtered using database information and optionally also allele frequency information. These germline filtering techniques are generally not as accurate as tumor normal subtraction. When using databases only to subtract germline variants, the TMB may be slightly higher than the more accurate T/N estimate. When using database and allele frequency information to remove germline variants, the TMB may be slightly underestimated for high purity tumor samples.

DRAGEN TMB comprise the following steps:

1. Variant calling

Please refer to "Somatic mode" for detailed variant calling options.

2. Eligible region detection

The user specifies regions of interests (eg coding regions) with qc-coverage-region1. The subset of these regions that also exceed the specified minimum required coverage vc-callability-tumor-thresh are identified as eligible.

3. Variant filters

The following variants are excluded from the TMB calculation:

• Non-PASS 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.
• 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. The tumor driver mutations filter relies on Nirvana annotations and will additionally require settings for --enable-variant-annotation=true, --variant-annotation-assembly, and --variant-annotation-data.
4. Support for germline variants

By default, germline variants are not counted towards TMB. Variants are determined as germline based on a database or a proxi filter. The database germline filter can be disabled with tmb-skip-db-filter. Disabling the database germline filter will effectively also disable the germline proxi filter.

Variants with a population allele count ≥ 50 that are observed in either the 1000 Genome or gnomAD database will be marked as germline. Use germline-tagging-db-threshold to change the population allele counts. The database germline filter relies on Nirvana annotations and requires settings for --enable-variant-annotation=true, --variant-annotation-assembly, and --variant-annotation-data.

• Proxi filter can be enabled with tmb-enable-proxi-filter. The proxi filter will flag any variants with VAF > 0.9 as germline. The proxi filter scans the variants surround a specific variant and identifies those variants with similar VAFs. The proxi window size that determines the number of surrounding variants can be specified with tmb-proxi-window-size. If 95% (default value for tmb-proxi-fraction-threshold) and no less than 5 (tmb-proxi-count-threshold) of the surrounding variants of similar VAF are germline, then mark the current variant also as germline.
• Proxi filter can also be done via a probabilistic approach, which can be enabled with tmb-enable-prob-proxi. It estimates the expected germline allele frequency using the surrounding germline variants and then tests whether the allele frequency of the target variant is similar to the expected germline allele frequency or not. P value threshold can be set by tmb-prob-proxi-p-value (the default value 1e-15 is set for ultra-deep sequenced samples, eg cfDNA)
• Note that proxi filters can be too aggressive for 100% pure cell lines. Probabilistic proxi filter can be problematic for mixing or contaminated samples, as these samples do not have clear germline variant allele frequency distributions.

When processing ctDNA samples it may be beneficial to also remove CH (clonal hematopoiesis) variants. Circulating tumor DNA generally has shorter fragment size. CH variants can be identified based on the insert size of the reads supporting the call. To capture the insert-size distribution for each variant call, it is required to specify vc-log-insert-size during variant calling (step1). Once specified, potential CH variants based on insert size distribution will be labeled in the output. Additional, CH variants can be also labeled via a bed file supplied to tmb-ch-bed. Variants other than germlines overlapping the region will be labeled as CH.

5. Support Nonsynonymous variants.

Nonsynonymous consequences are detected based on the Nirvana annotations. Nirvana variants that are annotated with the following consequences are labeled as nonsynonymous:

• 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

TMB emits a tmb.trace.csv file with detailed information on each variant used the TMB score. The trace file contains a column "Nonsynonymous" that indicates the appropriate status for each variant.

The subset of filtered variants that are nonsynonymous are used as numerator in the "Filtered Nonsyn Variant Count" metric.

6. TMB calculation

TMB = Filtered Variants / Eligible Region (Mbp)

Nonsynonymous TMB = Filtered Nonsynonymous Variants / Eligible Region (Mbp)

7. Maximum somatic allele frequency (MSAF)

The maximum somatic allele frequency (MSAF) outputs the estimated maximum somatic allele frequency of the sample. This is done via finding the confident somatic variants with highest allele frequency. MSAF is a rough approximate to the tumor fraction of cfDNA in peripheral blood samples. The MSAF mode can be enabled with tmb-enable-msaf.

Required

Command-line Option

Description

--enable-tmb true

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

--qc-coverage-tag-1

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

--qc-coverage-region-1

Specify the coding regions to use.

--vc-callability-tumor-thresh

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

--qc-coverage-reports-1=callability

The callability report is required for DRAGEN TMB.

--enable-variant-annotation=true

--variant-annotation-assembly

--variant-annotation-data

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

Recommended

Setting

Description

Tumor-Normal Panel/WES/WGS

Tumor-Only Panel/WES/WGS

Hight coverage bTMB

--vc-callability-tumor-thresh

The minimum coverage for usable coding regions

50

50

1000

--tmb-vaf-threshold

Variant mininum allele frequency for usable variants

0.05

0.05

0.002

--tmb-cosmic-count-threshold

Number of observations in cosmic for variant to be considered a driver mutation.

50

50

50

--tmb-skip-db-filter

Do not use Nirvana database to filter germline variants

TRUE

FALSE

FALSE

--tmb-enable-proxi-filter

Use allele frequency information to filter germline variants

OPTIONAL (Default is FALSE)

FALSE

TRUE

Optional

Command-line Option

Description

--tmb-vaf-threshold

Specify the minimum VAF threshold for a variant. Variants that do not meet the threshold are filtered out (default=0.05)

--tmb-cosmic-count-threshold

The minimum number of observations in cosmic for variant to be considered a driver mutation. Driver mutations are not counted in TMB. This setting has very little impact on WES/WGS, but can help avoid bias in small panels (default=50)

--tmb-skip-db-filter

Skip database germline filtering. The database germline filter is required for tumor-only samples, but can be skipped for tumor-normal (default=false)

--germline-tagging-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 (default=50)

--tmb-enable-proxi-filter

Enable proxi filter functionality in germline filtering. This is an optional feature that may be appropriate for T/O runs. In T/O mode the DB germline filter may not able to detect all germline variants, especially for ethnicity groups that are not well represented in germline databases. The proxi filter uses allele frequency information to help remove germline variants missed by the DB, and can help to obtain more accurate (lower) TMB values on samples with low tumor purity. In samples with high tumor purity this filter may be too aggressive and mark some somatic variants as germline resulting in too low TMB scores. (default=false)

--tmb-proxi-count-threshold

Proxi filter surrounding variant count threshold in germline filtering (default=5)

--tmb-proxi-fraction-threshold

Proxi filter surrounding variant db filter fraction threshold in germline filtering (default=0.95)

--tmb-proxi-window-size

Number of surrounding variants before and after the target variant for proxi filter (default=500)

--tmb-ch-bed

Variants in the region will be labeled as clonal hematopoiesis (CH) variants

--tmb-ch-insert-p-value

Minimum P value to classify a variant as CH using insert size (default=0.1)

--tmb-ch-insert-min-len

Minimum fragment size to test for CH using insert size (default=100)

--tmb-ch-insert-max-len

Maximum fragment size to test for CH using insert size (default=200)

--tmb-ch-insert-min-num

Minimum number of fragment size record to test for CH using insert size (default=50)

--tmb-enable-msaf

Enable MSAF output (default=false)

--tmb-msaf-p-value

Maximum P value (from insert size) to call confident somatic variant (default=1e-5)

--tmb-msaf-rank-num

If no confident somatic variant found, it will use the specified ranked variant (default=4)

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.

The TMB module also emits a tmb.trace.csv file that provides detailed information on each variant that was included in the TMB calculation.

When enabling MSAF, the information is output to <output prefix>.tmb.msaf.csv.