DNA Output

Refer to DNA Analysis Methods for more information.

Small Variant gVCF

File name: {SAMPLE_ID}_hard-filtered.gvcf.gz

The small variant genome variant call file contains information on all candidate small variants evaluated, including complex variants up to 15 bp from phased variant calling across the entire TSO 500 panel.

The variant status is determined by the FILTER column in the genome VCF as follows.

Filter	Note
PASS	PASS variants.
base_quality	Site filtered because median base quality of alt reads at this locus does not meet threshold.
filtered_reads	Site filtered because the fraction of reads is too large.
fragment_length	Site filtered because absolute difference between the median fragment length of alt reads and median fragment length of ref reads at this locus exceeds threshold.
low_depth	Site filtered because the read depth is too low.
low_frac_info_reads	Site filtered because the fraction of informative reads is below threshold.
long_indel	Site filtered because the indel length is too long.
mapping_quality	Site filtered because median mapping quality of alt reads at this locus does not meet threshold.
multiallelic	Site filtered because more than two alt alleles pass tumor LOD.
no_reliable_supporting_read	Site filtered because no reliable supporting somatic read exists.
read_position	Site filtered because median of distances between start/end of read and this locus is below threshold.
str_contraction	Site filtered due to suspected PCR error where the alt allele is one repeat unit less than the reference.
too_few_supporting_reads	Site filtered because there are too few supporting reads in the tumor sample.
weak_evidence	Somatic variant score (SQ) does not meet threshold.
systematic_noise	Site filtered based on evidence of systematic noise in normal sample.
excluded_regions	Site overlaps with VC excluded regions bed.

Small Variant Annotated JSON

File name: {SAMPLE_ID}_DNAVariants_Annotated.json.gz

The small variants annotated file provides variant annotation information for all nonreference positions from the genome VCF including pass and nonpass variants.

TMB Trace

The TMB trace file provides comprehensive information on how the TMB value is calculated for a given sample. All passing small variants from the small variant filtering step are included in this file. To calculate the numerator of the TmbPerMb value in the TMB JSON, set the TSV file filter to use the IncludedInTMBNumerator with a value of True.

The TMB trace file is not intended to be used for variant inspections. The filtering statuses are exclusively set for TMB calculation purposes. Setting a filter does not translate into the classification of a variant as somatic or germline.

Column	Description
Chromosome	Chromosome
Position	Position of variant
RefCall	Reference base
AltCall	Alternate base
VAF	Variant allele frequency
Depth	Coverage of position
CytoBand	Cytoband of variant
GeneName	Name of gene if applicable. A semicolon delimited list is used for multiple genes.
VariantType	Type of the variant: SNV, insertion, deletion, MNV
CosmicIDs	Cosmic IDs, if multiple concatenated by “;”
MaxCosmicCount	Maximum Cosmic study count
AlleleCountsGnomadExome	Variant allele count in gnomAD exome database
AlleleCountsGnomadGenome	Variant allele count in gnomAD genome database
AlleleCounts1000Genomes	Variant allele count in 1000 genomes database
MaxDatabaseAlleleCounts	Maximum variant allele count over the three databases
GermlineFilterDatabase	TRUE if variant was filtered by the database filter
GermlineFilterProxi	TRUE if variant was filtered by the proxi filter
CodingVariant	TRUE if variant is in the coding region
Nonsynonymous	TRUE if variant has any transcript annotations with nonsynonymous consequences
IncludedinTMBNumberator	TRUE if variant is used in the TMB calculation

Copy Number VCF

The copy number VCF file contains CNV calls for DNA libraries of the amplification genes targeted by DRAGEN TruSight Oncology 500 Analysis Software. The CNV call indicates fold change results for each gene classified as reference, deletion, or amplification.

The value in the QUAL column of the VCF is a Phred transformation of the p-value where Q=-10xlog10(p-value). The p-value is derived from the t-test between the fold change of the gene against the rest of the genome. Higher Q-scores indicate higher confidence in the CNV call.

In the VCF notation, <DUP> indicates the detected fold change (FC) is greater than a predefined amplification cutoff. <DEL> indicates the detected FC is less than a predefined deletion cutoff for that gene. This cutoff can vary from gene to gene.

In analysis versions prior to v2.5, <DEL> calls in the VCF are marked as LowValidation. The LowValidation filter indicates that the calls have been validated only with in silico data sets and are provided as information only.

Each copy number variant is reported as a fold change on normalized read depth in a testing sample relative to the normalized read depth in diploid genomes. Given tumor purity, you can infer the ploidy of a gene in the sample from the reported fold change.

Given tumor purity X%, for a reported fold change Y, you can calculate the copy number n using the following equation:

$$n=[(200Y)-2(100-X)]/X$$

For example, a tumor purity at 30% and a MET with fold change of 2.2x indicates that 10 copies of MET DNA are observed.