File name: {SampleID}_CombinedVariantOutput.tsv

The combined variant file contains the variants and biomarkers in a single file. The output contains the following variant types and biomarkers:

• Small variants (including EGFR complex variants)

• Copy number variants

• Tumor Mutational Burden (TMB)

• MSI

• DNA Fusions

The combined variant output file also contains Analysis Details and Sequencing Run Details sections. The details of each are listed in the following table:

Variant Filtering Rules

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 ctDNA.

• 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 and which have a canonical RefSeq transcript are present in the combined variant output.

  • Gene and transcript information is only present for variants belonging to canonical transcripts that are within the Gene 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> .

  • Gene is part of the copy number variant gene list

• 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.

  • Genes separated by a dash (-) indicate that the fusion directionality could be determined. Genes separated by a slash (/) indicate that the fusion directionality could not be determined.

The gene and exon coverage report files are tab-separated value (TSV) files with coverage values matching respectively the exons and genes specified in the manifest file.

Refer to DNA Analysis Methods for more information.

Small Variant Genome VCF

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

The small variant genome VCF file includes the variant call status for all targeted intervals, left-padded by 25 bp.

The Epidermal Growth Factor Receptor (EGFR) complex variant VCF includes phased EGFR variants. The FILTER column in the genome VCF determines the variant status. Refer to the following table for more information.

¹ Etc refers to other variant types not mentioned in the table.

² Reference positions and nonpassing variants with coverage below 1000X directly translate into low_depth. For variant calls, low_depth is not applied when a position has a PASS filter.

³ This is a static list of regions compiled by Illumina. Email Illumina Technical Support for more information.

Small Variant Annotated JSON

File name: {SAMPLE_ID}_DNAVariants_Annotated.json.gz

The small variants annotated file provides variant annotation information for all non-reference positions in the VCF, which includes non-pass variants. The variant consequence definition is available on the Sequence Ontology website.

All pass variant calls are annotated using the Illumina Annotation Engine (IAE), also known as Nirvana, with the following information (using the RefSeq transcript):

• HGNC Gene

  • Transcript

  • Exon

  • Consequence

  • c.HGVS

  • p.HGVS

• COSMIC

TMB Trace

File name: {Sample_ID}.tmb.metrics.csv

The TMB metrics file contains the tumor mutational burden metrics for each DNA sample. The file format uses the following CSV column convention, similar to other metric CSV files.

Sample Analysis Results JSON

File name: {SampleID}_SampleAnalysisResults.json

The sample analysis results file (SARJ) is an aggregated results file created for each sample. The SARJ file is used for the generation of downstream outputs. The file contains passing variants and passing variant annotations.

Copy Number Variants VCF

File name: {Sample_ID}_cnv.vcf

The copy number variants (CNV) file contains calls for DNA libraries of the CNV genes targeted by TruSight Oncology 500 ctDNA v2 and v1 assays. The CNV call indicates fold change results for each gene classified as reference, deletion, or amplification.

The value in the QUAL column of the copy number variants VCF is a Phred transformation of the p-value represented by the following equation:\

The p-value is derived from the t-test between the fold change (FC) 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 FC is greater than a predefined amplification cutoff. <DEL> indicates the FC is less than a predefined deletion cutoff for that gene. This cutoff can vary from gene to gene.

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

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

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

Copy Number Variants Metrics CSV

Copy number variant metrics are reported on a per sample level.

• Sex Genotyper—The predicted sex of the sample.

• Number of alignment records—The number of alignment records in the sample.

• Bases in reference genome—The number of bases in the reference genome.

• Average alignment coverage over genome—The average alignment coverage across the reference genome.

• Number of filtered records (total)—The number of total filtered records.

• Number of filtered records (duplicates)—The number of duplicated filtered records.

• Number of filtered records (MAPQ)—The number of MAPQ filtered records.

• Number of filtered records (unmapped)—The number of unmapped filtered records.

• Number of target intervals—The number of target intervals in the sample.

• Number of segments—The number of segments in the sample. Applicable only to CNV SLM mode.

• Number of amplifications—The number of amplifications in the sample. Applicable only to CNV SLM mode.

• Number of deletions—The number of deletions in the sample. Applicable only to CNV SLM mode.

• Number of passing amplifications—The number of passing amplifications in the sample. Applicable only to CNV SLM mode.

• Number of passing deletions—The number of passing deletions in the sample. Applicable only to CNV SLM mode.

Small Variant VCF

File name: {SampleID}_hard-filtered.vcf

The small variant file contains both phased variants and all other small variants. The header sections from both the phased variant (complex) VCF and the small variant VCF are included in this merged VCF. Variants that are found for both phased variants and small variants are only displayed as phased variants.

Fusions CSV

File name: {Sample_ID}_Fusions.csv

The fusions file contains all candidate fusions identified by the analysis pipeline.

The fusion columns are described in the following table. If you use Microsoft Excel to view this file, genes that are convertible to dates (for example, MARCH1) automatically convert to dd‑mm format (1-Mar).

The following table lists the meaning of the values in the direction column. The values are in the format used by Samtools.

When the analysis run completes, the DRAGEN TruSight Oncology 500 ctDNA 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.

Analysis Output Folder Structure

Single output folder structure is as follows.

• Logs_Intermediates

  • AdditionalSarjMetrics

  • Annotation—Contains outputs for small variant annotation.

    • Subfolders per sample ID—Contains the aligned small variants JSON.

  • CombinedVariantOutput

    • Subfolders per sample ID—Contains the combined variant output TSV files.

    • A combined output log file.

  • Contamination

    • Subfolders per sample ID—Contains the contamination metrics JSON file and output logs.

  • CoverageReports

  • DnaFusionFiltering

  • DragenCaller

    • Subfolders per sample ID—Contains the aligned BAM and index files, small variant VCF and gVCF, copy number variant VCF, MSI JSON, exon coverage report bed, and QC outputs in CSV format.

  • FastqValidation—Contains the FASTQ validation output log for the samples.

  • FastqGeneration

  • MetricsOutput

    • Subfolders per sample ID—Contains the metrics output TSV files.

    • A combined output log file.

  • ResourceVerification—Contains the resource file checksum verification logs.

  • Run QC—Contains the Run QC metrics JSON, Intermediate Run QC metrics JSON, and log file.

  • SampleAnalysisResults

    • Subfolders per sample ID—Contains the Sample Analysis Results JSON and detailed log file.

  • SampleSheetValidation—Contains the Intermediate sample sheet and validation log.

  • Passing Sample Steps - JSON file that contains the steps passed for each Sample ID

  • Tmb

    • Subfolders per sample ID—Contains the TMB metrics CSV, TMB trace TSV, and related files and logs. 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.

• Results

  • Metrics Output TSV (all Sample IDs)

  • Sample ID—The following outputs are produced for each sample:

    • Combined Variant Output TSV

    • Metrics Output TSV

    • TMB Trace TSV

    • Small Variant Genome VCF

    • Small Variant VCF

    • Small Variant Annotated JSON

    • Copy Number Variant VCF

    • MSI JSON

    • Fusions CSV

    • Exon Coverage Report TSV

    • Gene Coverage Report TSV

ICA Output Folder Structure

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.

High-Level Folder Structure

• Run ID

  • TSO500_Nextflow_logs

  • _manifest.json

  • Results

    • _tags.json

  • Logs_intermediates

  • Errors—This folder is only present when analysis fails

TSO500_Nextflow_logs Folder Structure

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

Results Folder Structure

Contains the aggregated MetricsOutput.tsv file at the root level. Additionally, the Results folder contains a subfolder for each sample ID.

• Results

  • MetricsOutput.tsv

  • Sample_1

  • Sample_2

  • Sample_<#>

The Results subfolder contains the following files:

• Results

  • MetricsOutput.tsv

  • <Sample_id>

    • CombinedVariantOutput.tsv

    • Fusions.csv

    • tmb.trace.tsv

    • hard-filtered.gvcf

    • hard-filtered.vcf

    • SmallVariants_Annotated.json.gz

    • cnv.vcf

    • exon_cov_report.tsv

    • gene_cov_report.tsv

    • MetricsOutput.tsv

    • microsat_output.json

Logs_intermediates Folder Structure

Contains folders for each submodule in the DRAGEN TSO 500 ctDNA 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

  • AdditionalSarjMetrics

  • Annotation

  • CombinedVariantOutput

  • Contamination

  • CoverageReports

  • DnaFusionFiltering

  • DragenCaller

  • FastqValidation

  • FastqGeneration

  • MetricsOutput

  • PassingSampleSteps

  • ResourceVerification

  • Run QC

  • SampleAnalysisResults

  • SampleSheetValidation

  • Tmb

All logs in Logs_Intermediates are generated from the running analysis software. Inputs to the running Docker container (for example, the run folder, sample sheet, and FASTQ folder) are mapped from native locations on the server to the following locations in the container:

The paths in the log messages refer to paths within the running docker container, not paths on the server.

Errors Folder Structure

Contains Errors.tsv. This file contains the summary of all the errors encountered during pipeline execution.

• Errors

  • Errors.tsv

NovaSeq 6000Dx Analysis Application Output Folder Structure

The following files and folders are created during analysis by NovaSeq 6000Dx Analysis Application:

• analysisResults.json

• CopyComplete.txt

• edgeos.nextflow.config

• inputs/

  • sampleMapping.json

  • SampleSheet.csv

  • SampleSheet.json

• Logs_Intermediates

• Manifest.tsv

• params.json

• Results/

• workflowLogs/

  • nf-main-***.log

When the analysis run completes, the analysis application generates an analysis output in a specified location. To view analysis output, follow the steps below:

1. On the “Completed” runs tab, select the run

2. Review the run details page, and this will give the information to access the output folder

3. External Location: is the input for the run

4. Analysis Output Folder: is where the output is stored. To navigate to this page, follow the “server location” and the gds analysis output folder

5. Navigate to the directory that contains the analysis output folder

6. Open the folder, and then select the files that you want to view

File Name: MetricsOutput.tsv

The metrics output file is a final combined metrics report that provides sample status, key analysis metrics, and metadata in a tab-separated values (TSV) file. Sample metrics within the report indicate guideline‑suggested lower specification limits (LSL) and upper specification limits (USL) for each sample in the run.

One metrics output file is generated for the entire run. An additional file is generated for each sample

Run Metrics

Run metrics from the analysis module indicate the quality of the sequencing run.

Review the following metrics to assess run data quality:

The values in the Run Metrics section are listed as NA in the following situations:

• The analysis was started from FASTQ files.

• The analysis was started from BCL files and the InterOp files are missing or corrupt.

• [NovaSeqX Plus only] There is no PCT_PF_READS value in NovaSeqX Plus runs, so the PCT_PF_READS value will always be NA.

Sample QC Metrics

Review the following metrics to assess sample data quality:

*The recommended threshold of 0.059 for GENE_SCALED_MAD only applies to real cell‑free DNA.

DNA expanded metrics are provided for information only. They can be informative for troubleshooting but are provided without explicit specification limits and are not directly used for sample quality control. For additional guidance, contact Illumina Technical Support.

The following table lists the genes that have associated block listed sites. For the exact location of the block listed site, contact Illumina Technical Support.

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