Refer to DNA Analysis Methods for more information.
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.
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.
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.
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:
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.
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.
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
IncludedinTMBNumerator
TRUE if variant is used in the TMB calculation
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.
Chromosome
Chromosome
Position
Position of variant
RefCall
Reference base
AltCall
Alternate base
VAF
Variant allele frequency
Depth
Coverage of position
long_indel
One metrics output file is generated for the entire run. An additional file is generated for each sample (or DNA-RNA pair).
The MetricsOutput.tsv file contains the following quality control metrics for all samples:
DNA library QC metrics for:
Small variant calling
TMB
MSI
CNV
RNA library QC metrics
Run QC metrics, analysis status, and contamination
This TSV file also includes expanded DNA library QC metrics per sample, based on total reads, collapsed reads, chimeric reads, and on-target reads. Analysis using RNA samples also produces RNA library QC metrics and expanded RNA library QC metrics per sample based on total reads and coverage.
The MetricsOutput.tsv file is a final combined metrics report with sample status, key analysis metrics, and metadata. Sample metrics within the report include suggested lower specification limits (LSL) and upper specification limits (USL) for each sample in the run.
For troubleshooting information, refer to
[HRD] GIS
The Illumina DRAGEN TruSight Oncology 500 Analysis Software allows for analysis of sequencing data generated from the TruSight Oncology 500 HRD assay. When HRD samples are analyzed new results and metrics are included in the CombinedVariantOutput and MetricsOutput files respectively. The following tables detail how these scores and QC metrics are derived.
Genomic Instability Score (GIS)
Proprietary Genomic Instability Score (GIS) indicating level of genomic instability in sample genome. Combination of Loss of Heterozygosity (LOH), Telomeric allelic imbalance and Large-scale State Transitions (LST) scores. The GIS scores provided by TruSight Oncology 500 HRD show good correlation (R2= 0.98) with Myriad Genetics GIS however they are not identical (Refer to TruSight Oncology 500 HRD Product Data Sheet Doc# M-GL-00748 for more details). GIS from alternative HRD assays should be not be considered equivalent to Illumina/Myriad GIS.
The GIS algorithm within the TSO500 pipeline (which does not have a cell line mode due to the TSO500 pipeline being non-configurable) is only intended for FFPE samples. Cell line samples will not accurately report GIS results as the tumor fraction (>90%) is too high to reliably distinguish tumor vs germline variants.
File name: {Pair_ID}_CombinedVariantOutput.tsv
The combined variant output file contains the variants and biomarkers in a single file that is based on a single sample. If using pair ID, the file is based on paired DNA and RNA samples from the same individual. The output contains the following variant types and biomarkers:
Small variants
Copy number variants (CNV) (with absolute copy number when HRD Assay is run)
PCT_TARGET_HRD_50X
Percent of HRD probe SNP panel covered by at least 50X coverage
DNA Library QC Metrics for GIS
EXCESSIVE_TF
EXCESSIVE TF indicates if there is excessive tumor content in sample. Troubleshooting: Samples with pure tumor fraction >90% are outside the design for GIS estimation (this includes pure tumor cell lines)
DNA Library QC Metrics for GIS
ALLELE_DOSAGE_RATIO
Proprietary Myriad Genetics estimate of b-allele dosage based on b-allele noise/signal ratio. B-Allele noise is correlated with coverage; lower coverage samples will have higher noise. B-allele signal is also correlated with tumor fraction; a higher tumor fraction produces a higher signal for b-allele sites. Samples with lower tumor fraction and higher amount of noise (or lower coverage) will have higher Allele Dosage Ratio. The upper limit of the score is 50, therefore any sample with 50 Allele Dosage Ratio can be assumed to have tumor fraction close to zero and typically has a GIS = 0.
DNA Expanded Metrics
MEDIAN_TARGET_HRD_COVERAGE
Median target fragment coverage across all target positions in the genome. Coverage is the total number of non-duplicate pair alignments that overlap.
DNA Expanded Metrics
TMB
MSI
Fusions
Splice variants
GIS (when HRD Assay is run)
Gene-level Loss of Heterozygosity (when HRD Assay is run)
Exon-level CNVs
The combined variant output file also contains Analysis Details and Sequencing Run Details sections. The details of each are listed in the following table:
- Pair ID - DNA sample ID (if DNA is run) - RNA sample ID (if RNA is run) - Output date - Output time - Module version - Pipeline version (Docker image version #)
- Run name - Run date - DNA sample index ID (if DNA is run) - RNA sample index ID (if RNA is run) - [HRD] Sample feature - Instrument ID - Instrument control software version - Instrument type - RTA version - Reagent cartridge lot number
Combined variant output produces small variants with blank fields in the following situations:
The variant has been matched to a canonical RefSeq transcript on an overlapping gene not targeted by TruSight Oncology 500.
The variant is located in a region designated iSNP, indel, or Flanking in the TST500_Manifest.bed file located in the Resources folder.
Small Variants - All variants with the FILTER field marked as PASS in the hard-filtered genome VCF are present in the combined variant output.
Gene information is only present for variants belonging to canonical transcripts that are within the Gene Allow List–Small Variants.
Transcript information is only present for variants belonging to canonical transcripts that are within the Gene Allow List–Small Variants.
Copy Number Variants - Copy number variants must meet the following conditions:
FILTER field marked as PASS.
ALT field is <DUP or <DEL> .
Fusion Variants - Fusion variants must meet the following conditions:
Passing variant call (KeepFusion field is true).
Contains at least one gene on the fusion allow list.
Biomarkers TMB/MSI - Always present when DNA sample is processed.
Splice Variants - Passing splice variants that are contained on genes EGFR, MET, and AR.
Biomarker GIS - Present only if TruSight Oncology 500 HRD analysis is performed
Loss of Heterozygosity - Present only when TruSight Oncology 500 HRD is run. Loss of heterozygosity (LOH) must meet the following condition:
MCN field is equal to 0
Exon-level CNVs - Exon-levels CNVs must meet the following conditions:
BRCA1 or BRCA2 contains at least one affected exon.
ALT field is <DUP> or <LOSS> .
When the analysis run completes, the DRAGEN TruSight Oncology 500 Analysis Software generates an analysis output folder in a specified location.
To view analysis output, navigate to the analysis output folder and select the files that you want to view.
Single output folder structure is as follows.
Logs_Intermediates
AdditionalSarjMetrics— Contains per pair ID calculations to support the PCT_TARGET_250X metric.
Annotation—Contains outputs for small variant annotation.
Subfolders per sample ID—Contains the aligned small variants JSON.
Results
Metrics Output TSV (all pair IDs)
Pair ID—The following outputs are produced for each sample:
Multiple output folder structure is as follows.
Demultiplex Output
A Logs_Intermediates folder containing FASTQ files per sample.
Node(X) Output—The following outputs are produced for each node used:
This section describes each output folder generated during analysis and where to find metric and analytic files when the pipeline is executed. The same output folder structure and content exist in ICA and BaseSpace Sequence Hub.
Run ID
TSO500_Nextflow_logs
_manifest.json
The TSO_500_Nextflow_Logs provides information related to the execution of the pipeline on ICA as a whole and for specific nodes (when an analysis is split across multiple nodes). It contains files used to execute parts of the workflow on different nodes as well as records of the nextflow execution on those nodes.
TSO_500_Nextflow_Logs
_manifest.json
Contains the aggregated MetricsOutput.tsv file at the root level. Additionally, the Results folder contains a subfolder for each pair ID.
Results
MetricsOutput.tsv
Sample_1
The Results subfolder contains the following files:
Results
MetricsOutput.tsv
<Pair_id>
Contains folders for each submodule in the DRAGEN TSO 500 on ICA pipeline. The folders contain a copy of all the relevant files required to create the metric output files and report files, as well as the combined log files at the root level and subfolders for each sample.
Logs_intermediates
DnaDragenCaller
AdditionalSarjMetrics
Contains Errors.tsv. This file contains the summary of all the errors encountered during pipeline execution.
Errors
Errors.tsv
CombinedVariantOutput
Subfolders per pair ID—Contains the combined variant output TSV files.
A combined output log file.
Contamination
Subfolders per DNA sample ID—Contains the contamination metrics JSON file and output logs.
DnaDragenCaller
Subfolders per sample ID—Contains the aligned BAM and index files, small variant VCF and gVCF, copy number variant VCF, MSI JSON, and QC outputs in CSV format.
DnaDragenExonCNVCaller
Subfolders per DNA sample ID—Contains the exon-level CNV JSON,the supporting calculation, and the QC files.
DnaFastqValidation—Contains the FASTQ validation output log for DNA samples.
FastqDownsample
Subfolders per RNA sample ID—Contains FASTQ files and output logs.
FastqDownsample output
FastqGeneration
Gis—Contains GIS-related files for HRD samples.
Subfolders per HRD sample ID—Contains the GIS JSON, the supporting calculation, and the QC files.
Also contains the annotated CNV VCF and gene level TSV file with absolute copy number and minor copy number information
LrAnnotation
Subfolders per DNA sample ID—Contains the annotated exon-level CNV JSON.
LrCalculator
Subfolders per DNA sample ID—Contains the exon-level CNV VCF.
MetricsOutput
Subfolders per pair ID—Contains the metrics output TSV files.
A combined output log file.
ResourceVerification—Contains the resource file checksum verification logs.
RnaAnnotation
Subfolders per RNA sample ID—Contains the annotated splice variant JSON.
RnaDragenCaller
Subfolders per sample ID—Contains the aligned BAM, fusion candidates CSV and QC outputs in CSV format.
RnaFastqValidation—Contains the FASTQ validation output log for RNA samples.
RnaFusion
Subfolders per RNA sample ID—Contains the All Fusions CSV and Fusion Processor logs.
RnaQcMetrics
Subfolders per RNA sample ID—Contains the RNA QC metrics JSON.
RnaSpliceVariantCalling
Subfolders per RNA sample ID—Contains the splice variants VCF.
Run QC—Contains the Run QC metrics JSON, Intermediate Run QC metrics JSON, and log file.
SampleAnalysisResults
Subfolders per pair ID—Contains the Sample Analysis Results JSON and detailed log file.
SampleSheetValidation—Contains the Intermediate sample sheet and validation log.
Tmb
Subfolders per DNA sample ID—Contains the TMB metrics CSV, TMB trace TSV, and related files and logs. passing_sample_steps.json
—Contains the steps passed for each sample ID.
pipeline_trace.txt—Contains a summary and troubleshooting file that lists each Nextflow task executed and the status (for example, COMPLETED or FAILED).
run.log—Contains a complete trace-level log file describing the Nextflow pipeline execution.
run_report.html—Contains high-level run statistics (performance, usage, etc.)
run_timeline.html —Contains timeline-related information about the analysis run.
Metrics Output TSV
TMB Trace TSV
Small Variant Genome VCF
Small Variant Genome Annotated JSON
Copy Number Variant VCF
GIS JSON
MSI JSON
Large Rearrangements CNV VCF
Large Rearrangements CNV Annotated JSON
All Fusion CSV
Splice Variant VCF
Splice Variant Annotated JSON
A Logs_Intermediates folder containing step specific and component specific outputs and logs for every step/component run in the analysis pipeline for the sample run on the node.
A Results folder containing results only for the sample run on the node.
Gathered Output
A Logs_Intermediates folder containing step specific and component specific outputs and logs for every step/component run in each analysis pipeline on every node—this contains outputs for all samples and pairs ran across all nodes in the analysis.
A Results folder containing results for all samples and pairs ran across all nodes—results are organized by Pair_ID, then Sample_ID. This folder also contains summary files which contain information on all samples.
_tags.json
Logs_intermediates
Errors—This folder is only present when analysis fails
Sample_<#>
_tags.json
<SampleName>_MetricsOutput.tsv
<DNA_Sample_id>
CopyNumberVariants.vcf
DNAMergedSmallVariants_Annotated.json.gz
MergedSmallVariants.genome.vcf
MergedSmallVariants.vcf
microstat_output.json
TMB_Trace.tsv
<RNA_Sample_id>
AllFusions.csv
RNA_Annotated.json.gz
SpliceVariants.vcf
FastqGeneration
MetricsOutput
DnaDragenExonCnvCaller
DnaFastqValidation
Gis
Tmb
SampleAnalysisResults
SampleSheetValidation
passing_sample_steps.json
RnaFusion
Contamination
Annotation
RnaAnnotation
RnaDragenCaller
RnaSpliceVariantCalling
RunQc
FastqDownsample
PassingSampleSteps
ResourceVerification
LrCalculator
LrAnnotation
RnaQcMetrics
RnaFastqValidation
Refer to RNA Analysis Methods for more information.
The splice variant VCF contains all candidate splice variants targeted by the analysis panel identified by the RNA analysis pipeline. You can apply the following filters for each variant call:
Refer to the headers in the output for more information about each column.
If available, each splice variant is annotated using the Illumina Annotation Engine. The following information is captured in the JSON:
HGNC Gene
Transcript
Exons
Introns
The all fusions CSV file contains all candidate fusions identified by the DRAGEN RNA pipeline. Two output columns in the file describe the candidate fusions: Filter and KeepFusion.
The following table describes the semicolon-separated output found in the Filter columns. The output is either a confidence filter or information only as indicated. If none of the confidence filters are triggered, the Filter column contains the output PASS, else it contains the output FAIL.
Filter Column Output
The KeepFusion column of the output has a value of TRUE when none of the confidence filters are triggered.
Refer to the headers in the output for more information about each column.
Fusion Columns
When using Microsoft Excel to view this report, genes that are convertible to dates (such as MARCH1 automatically convert to dd-mm format (1 Mar) by Excel. The following are fusion allow list genes:
ABL1
AKT3
ALK
AR
Canonical
Consequence
MIN_SUPPORT
Confidence filter
The fusion candidate has very few fusion supporting reads (< 5 supporting read pairs).
READ_THROUGH
Confidence filter
The breakpoints are cis neighbors (< 200 kbp) on the reference genome.
ANCHOR_SUPPORT
Information only
Read alignments of fusion supporting reads are not long enough (12 bp) at either of the two breakpoints.
HOMOLOGOUS
Information only
The candidate is likely a false candidate generated because the two genes involved have high gene homology.
LOW_ALT_TO_REF
Information only
The number of fusion supporting reads is < 1% of the number of reads supporting the reference transcript at either of the two breakpoints.
LOW_GENE_COVERAGE
Information only
Each breakpoint in an enriched gene has fewer than 125 bp with nonzero read coverage.
NO_COMPLETE_SPLIT_READS
Confidence filter
For every fusion-supporting split read, the total number of aligned bases across two breakpoints is less 60% of the read length.
UNENRICHED_GENE
Confidence filter
Neither of the two parent genes is in the enrichment panel.
Gene B Breakpoint
[Information only] The chromosome and offset of the Gene B side of the fusion.
Gene B Location
Location of the breakpoint within Gene B: - IntactExon—Matches exon boundary - BrokenExon—Inside an exon - Intronic—Within an intron - Intergenic—No gene overlap (currently excluded) If multiple genes in Gene B, then semicolon separated list of locations. This column is used internally to identify genes to report when a breakpoint occurs in a region overlapping multiple genes. Occasionally, additional values are listed for genes that were excluded from the GeneB list.
Gene B Sense
Boolean indicating whether left/right breakpoint order suggests fusion transcript is in the same sense of Gene B. If multiple genes are in Gene B, then semicolon separated list of bools.
Gene B Strand
Strand of Gene B, + for forward, - for reverse.
Score
The quality of fusion as determined by DRAGEN server.
Filter
The filter associated with the fusion as determined by the respective caller. Results from different callers are not equivalent.
Ref A Dedup
Gene A uniquely mapping reads paired across or split by the junction. Does not support fusion. Duplicate reads are not included.
Ref B Dedup
Gene B uniquely mapping reads paired across or split by the junction. Does not support fusion. Duplicate reads are not included.
Alt Split Dedup
Uniquely mapping reads split by the junction. Supports fusion. Duplicate reads are not included.
Alt Pair Dedup
Uniquely mapping reads paired across junction. Supports fusion. Duplicate reads are not included.
KeepFusion
The determination whether the fusion should be kept or dropped from the list of fusions.
Fusion Directionality Known
Whether fusion directionality is known and indicated by gene order.
AXL
BCL2
BRAF
BRCA1
BRCA2
CDK4
CSF1R
EGFR
EML4
ERBB2
ERG
ESR1
ETS1
ETV1
ETV4
ETV5
EWSR1
FGFR1
FGFR2
FGFR3
FGFR4
FLI1
FLT1
FLT3
JAK2
KDR
KIF5B
KIT
KMT2A
MET
MLLT3
MSH2
MYC
NOTCH1
NOTCH2
NOTCH3
NRG1
NTRK1
NTRK2
NTRK3
PAX3
PAX7
PDGFRA
PDGFRB
PIK3CA
PPARG
RAF1
RET
ROS1
RPS6KB1
TMPRSS2
LowQ
Splice variant score < passing quality score threshold value of 1.
PASS
Splice variant score ≥ passing quality score threshold value of 1.
LowUniqueAlignments
All splice junction supporting reads map to a unique genomic interval near at least one of the two splice sites.
DOUBLE_BROKEN_EXON
Confidence filter
If both breakpoints are distant from annotated exon boundaries, the number of supporting reads do not satisfy a high threshold requirement (≥ 10 supporting reads).
LOW_MAPQ
Confidence filter
All fusion supporting read alignments at either of the breakpoints have MAPQ < 20.
LOW_UNIQUE_ALIGNMENTS
Confidence filter
All fusion supporting read alignments map to a unique genomic interval at either of the breakpoints.
LOW_SCORE
Confidence filter
Gene A
The gene associated with the A side of the fusion. A semicolon delimited list is used for multiple genes.
Gene B
The gene associated with the B side of the fusion. A semicolon delimited list is used for multiple genes.
Gene A Breakpoint
[Information only] The chromosome and offset of the Gene A side of the fusion.
Gene A Location
Location of the breakpoint within Gene A: - IntactExon—Matches exon boundary - BrokenExon—Inside an exon - Intronic—Within an intron - Intergenic—No gene overlap (currently excluded) If multiple genes are in Gene A, then semicolon separated list of locations. This column is used internally to identify genes to report when a breakpoint occurs in a region overlapping multiple genes. Occasionally, additional values are listed for genes that were excluded from the GeneA list.
Gene A Sense
Boolean indicating whether left/right breakpoint order suggests fusion transcript is in the same sense of Gene A. If multiple genes are in Gene A, then semicolon separated list of bools.
Gene A Strand
Strand of Gene A, + for forward, - for reverse.
The fusion candidate has probabilistic score as determined by the features of the candidate.