DRAGEN TruSight™ Oncology 500 and DRAGEN TruSight™ Oncology 500 ctDNA Analysis Software support secondary analysis for the data generated by assays in the TruSight™ Oncology 500 (TSO 500) portfolio:

• DRAGEN TruSight™ Oncology 500 Analysis Software supports the following assays, all Research Use Only (RUO):

  • TruSight Oncology 500 Assay

  • TruSight Oncology 500 High-Throughput Assay

  • TruSight Oncology 500 HRD Add-on Assay

  • TruSight Oncology 500 v2 Assay

• DRAGEN TruSight™ Oncology 500 ctDNA Analysis Software supports the following assays, all Research Use Only (RUO):

  • TruSight Oncology 500 ctDNA Assay v1

  • TruSight Oncology 500 ctDNA v2 Assay

Depending on the version, the analysis software is available on:

• Illumina Connected Analytics (ICA): A cloud-based secure platform for data analysis and management. Analysis setup, monitoring, and results access is facilitated via user-friendly interface of BaseSpace Sequence Hub.

• DRAGEN server: An on-premises server that offers secondary analysis in a fraction of the time compared with a traditional CPU-based system.

• On-board of select instruments: Enabled by a user-friendly analysis application installed on a sequencer.

This resource provides information on installation, configuration, running, troubleshooting as well as analysis algorithms of DRAGEN TruSight™ Oncology 500 and DRAGEN TruSight™ Oncology 500 ctDNA Analysis Software.

The following sections describe performance testing methods.

Analytical Performance Testing

Illumina tests the analytical performance of variant calling for TruSight Oncology 500 and TruSight Oncology 500 ctDNA assays using an approach that covers the entire workflow including library preparation, sequencing, and secondary analysis. This approach is used to test a diverse selection of variants. When the variant calling pipeline is expanded to call a new variant class, this approach is always used.

Some versions of the DRAGEN TruSight™ Oncology 500 Analysis Software and DRAGEN TruSight™ Oncology 500 ctDNA Analysis Software include results generated by features tested in silico and by beta features. Beta features have not been fully evaluated for performance, see Beta Features for more details.

In Silico Testing Methods

Illumina uses in silico testing to the test the ability of the software to call an expanded scope of clinically relevant variants, including rare variants. In silico testing is used as a complementary method to analytical performance testing with wet lab step to expand the scope of testing. For example, while Illumina has analytically verified the performance of the software for calling complex variants in EGFR, the in silico testing approach characterizes the ability of the software to call complex variants in other genes.

For in silico testing of the DRAGEN TruSight Oncology 500 Analysis Software, variants of interest are compiled from public databases like COSMIC and ClinVar. Each variant is simulated at different VAF levels by, depending on the variant class, spiking in mutant reads into a normal FFPE background (for sequence variants) or by increasing or decreasing the coverage of exons in the normal FFPE sample (for CNVs, for example, exon-level CNVs). The simulated reads match the expected quality of typical FFPE samples, such as fragment length, error rate, and family size. After the simulation, the software processes samples with spiked-in variants and determines the results. This approach does not include library prep and sequencing of tumor FFPE samples that include the rare variants of interest. The software reports these variants, but analytical verification was not performed.

In silico tested features for DRAGEN TruSight Oncology 500 Analysis Software and DRAGEN TruSight Oncology 500 ctDNA Analysis Software are summarized in the table below.

Beta Features

DRAGEN TruSight Oncology 500 Analysis Software also includes beta features. The features have not been verified by Illumina due to limited access to samples or lack of an appropriate orthogonal method to perform testing, and, the use of in silico testing alone was not sufficient for verification purposes.

Customers are responsible for evaluating and demonstrating performance of any beta features they choose to implement. Beta features are indicated as such in the CombinedVariantOutput.tsv file produced by the DRAGEN TruSight Oncology 500 Analysis Software. Illumina will continue to evaluate beta features with intent to fully release upon completion of verification for each feature.

Beta features are summarized in the table below.

The block list represents high noise regions in the panel where false positive variant calls are likely produced. As a result, all positions in the gVCF are marked as Filter=excluded_regions to indicate variant call results are not reliable in such regions.

The block list includes the following genes:

• HLA A

• HLA B

• HLA C

• KMT2B

• KMT2C

• KMT2D

• chrY

• Any position with VAF 1% occurrence in six or more of the 60 baseline samples.

The block list represents high noise regions in the panel where false positive variant calls are likely produced. As a result, all positions in the gVCF are marked as Filter=excluded_regions to indicate variant call results are not reliable in such regions.

The block list includes the following genes:

• HLA A

• HLA B

• HLA C

• KMT2B

• KMT2C

• KMT2D

• chrY

• Any position with VAF 1% occurrence in six or more of the 60 baseline samples.

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

Sequencing data stored in BCL format are demultiplexed through a process that uses the index sequences unique to each sample to assign clusters to the library from which they originated. Each cluster contains two indexes (i7 and i5 sequences, one at each end of the library fragment). The combination of those index sequences are used to demultiplex the pooled libraries.

After demultiplexing, this process generates FASTQ files, which contain the sequencing reads for each individual sample library and the associated quality scores for each base call, excluding reads from any clusters that did not pass filter.

Sequencing data stored in BCL format are demultiplexed through a process that uses the index sequences unique to each sample to assign clusters to the library from which they originated. Each cluster contains two indexes (i7 and i5 sequences, one at each end of the library fragment). The combination of those index sequences are used to demultiplex the pooled libraries.

After demultiplexing, this process generates FASTQ files, which contain the sequencing reads for each individual sample library and the associated quality scores for each base call, excluding reads from any clusters that did not pass filter.

When using the NovaSeq 6000Dx application, DRAGEN TSO 500 analysis starts automatically after sequencing completes.

In addition to TSO 500 managed sample sheet validations, ICA managed TSO 500 errors include the following:

DRAGEN TruSight Oncology 500 Analysis Software can be used to run in parallel on multiple DRAGEN servers to decrease overall processing time. This is possible using a three stage process called scatter/ gather, which consists of demultiplexing, analysis, and result gathering.

Demultiplex and Scatter

The first stage is demultiplexing. Demultiplexing runs once on the entire run folder, generates FASTQ files for each sample in the run, and then separates sample files into respective folders. Once complete, note the output directory containing the sample directories holding the FASTQ files.

The process for scattering the analysis on multiple DRAGEN servers is as follows:

Overview

A sample sheet is required for each analysis with DRAGEN TruSight Oncology 500 Analysis Software. A sample sheet is a comma-separated value (*.csv) file format used by Illumina instruments, platforms, and analysis pipelines to store settings and data for sequencing and analysis. The DRAGEN TruSight Oncology 500 Analysis Software is compatible with the sample sheet v2. For general information on the sample sheet v2, refer to .

The sample sheet includes a list of samples and their index sequences, along with additional information required to run DRAGEN TruSight Oncology 500 Analysis Software. For example, DNA samples with the TruSight Oncology 500 HRD add-on probes must be indicated in the Sample Feature column of the sample sheet. Appropriate index adapter sequences are determined by the assay used to perform analysis.

When running analysis on a standalone DRAGEN server or on ICA, a valid sample sheet can be created by:

When using BSSH Run Planning to generate a sample sheet to auto-launch analysis on ICA, you must designate "Start from Fastq" to be True or False (default is False). If you choose "False", BSSH will kick off the TSO 500 pipeline normally using BCL input from the run folder.

If you choose "True" for this option, BSSH will run two ICA pipelines in sequence:

• First, it will run the BCL Convert v3.10.9 pipeline to generate the FASTQ files

• Second, it will kick off the TSO 500 pipeline using the FASTQ files generated above as the input

The software processes sequencing data to perform quality control, detect variants, determine tumor mutational burden (TMB), microsatellite instability (MSI) status, and genomic instability score (GIS), and report results. The following sections describe the analysis methods used in DRAGEN TruSight Oncology 500 Analysis Software.

DRAGEN TruSight Oncology 500 Analysis Software uses the following workflows to analyze sequencing data.

• FASTQ Generation

• DNA Analysis

Overview

A sample sheet is required for each analysis with DRAGEN TruSight Oncology 500 Analysis Software. A sample sheet is a comma-separated value (*.csv) file format used by Illumina instruments, platforms, and analysis pipelines to store settings and data for sequencing and analysis. The DRAGEN TruSight Oncology 500 Analysis Software is compatible with the sample sheet v2. For general information on the sample sheet v2, refer to .

The sample sheet includes a list of samples and their index sequences, along with additional information required to run DRAGEN TruSight Oncology 500 Analysis Software. For example, DNA samples with the TruSight Oncology 500 HRD add-on probes must be indicated in the Sample Feature column of the sample sheet. Appropriate index adapter sequences are determined by the assay used to perform analysis.

When running analysis on a standalone DRAGEN server or on ICA, a valid sample sheet can be created by:

Metrics Output

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:

Prerequisites

Illumina Connected Analytics (ICA) subscription includes access to DRAGEN TruSight Oncology 500 ctDNA Analysis Software. To get started, you need:

• An ICA account with a valid subscription

Here are the articles in this section:

Prerequisites

Illumina Connected Analytics (ICA) subscription includes access to DRAGEN TruSight Oncology 500 Analysis Software. To get started, you need:

• An ICA account with a valid subscription

The block list represents high noise regions in the panel where false positive variant calls are likely produced. As a result, all positions in the gVCF are marked as Filter=excluded_regions to indicate variant call results are not reliable in such regions.

The block list includes the following genes:

• HLA A

• HLA B

• HLA C

Methods for Launching Analysis

Illumina Connected Analytics (ICA) supports the following methods for launching DRAGEN TruSight Oncology 500 Analysis Software.

• —Stream run data directly from the instrument to ICA via a specially configured sample sheet and automatically begin DRAGEN TSO 500 analysis.

Metrics Output

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:

Prerequisites

• A NovaSeq 6000Dx sequencing instrument with paired DRAGEN server v4

• Illumina Run Manager installed by Illumina support personnel

• TSO500Combined and TSO500_HRD licenses installed by Illumina support personnel

  • TSO500Combined and TSO500_HRD licenses are pre-installed in manufacturing since February 2025 and will need to be installed only for instruments purchased prior to that

• The user installing the app must have admin privileges on Illumina Run Manager

Installation Instructions

1. Contact Illumina Customer Care Contact Illumina Customer Care at to obtain installation package for Illumina DRAGEN TruSight Oncology 500 (HRD) Analysis Application on NovaSeq 6000Dx.

2. Download the installation package Download the installation package provided in the email by Illumina Customer Care. The link will expire after 7 days.

1. Installation package contents The installation package contains the following:

1. Install the DRAGEN version using the ires:

   1. Log into Illumina Run Manager as a user with admin credentials

   2. Navigate to the top left menu to "DRAGEN" in the drop down

   3. Select "Add DRAGEN Installer" and upload the DRAGEN ires file

\

The contamination score evaluates presence of sample-to-sample contamination. The algorithm uses common germline SNPs in the homozygous state expected to have variant allele frequencies (VAF) at 0% and 100%. In contaminated samples, the VAFs shift away from the expected values allowing the detection of sample-to-sample contamination.

Contamination Score Calculation

The contamination score is calculated using the SNP error file and Pileup file that are generated during the small variant calling, as well as the TMB trace file. The algorithm includes the following steps:

• All positions that overlap with a pre-defined set of common SNPs that have variant allele frequencies of < 25% or > 75% are collected (only SNP are considered, indels are excluded)

• Variants in CNV events are removed using a clustering method

• The likelihood that the positions are an error or a real mutation is calculated by:

  • Estimating the error rate per sample

CONTAMINATION_SCORE = sum(log10(P(v_i is False Positive)))

Contamination Score Interpretation

• The contamination score is output in the metrics output file, MetricsOutput.tsv

• If a contamination score is equal or below 1457 (the upper specification limit provided in the "USL Guideline" field in the metrics output file, see ), the sample has less than 2% sample-to-sample contamination.

• If a contamination score is above 1457, the sample has more than 2% sample-to-sample contamination. In this case, an estimation of the contamination can be obtained from the PCT_CONTAMINATION_EST metric, see more details on the . As noted, PCT_CONTAMINATION_EST is not valid unless the contamination score exceeds 1457.

• Visual examination can help determine if a shift of VAFs is due to true contamination

How to build a VAF plot for visual examination

1. To build a VAF plot, use the {Sample_ID}.tmb.trace.csv file. Filter to only germline variants (for example, by using tags "Germline_DB" and "Germline_Proxi" in the column "Status") and use values in the VAF column.

2. Select Scatter from the Charts menu

3. Review plot as described above analyzing whether variants are scattered or clustered around 50% and 100% VAF

Methods for Launching Analysis

Illumina Connected Analytics (ICA) supports the following methods for launching DRAGEN TruSight Oncology 500 Analysis Software.

• Auto-launch—Stream run data directly from the instrument to ICA via a specially configured sample sheet and automatically begin DRAGEN TSO 500 analysis.

• —Initiate DRAGEN TSO 500 analysis on ICA using the run files and sample sheet files in the project.

For more information about using ICA or BaseSpace Sequence Hub, refer to the following support pages on the Illumina support site.

Resources used by ICA Analyses

EC2 Instance Support

As of DRAGEN TSO500 v2.6.2, ICA pipelines are able to run on F2 instance type nodes (in addition to F1, when available).

For additional details on performance and availability of supported instance types, please see:

BCL Convert in parallel by lane

As of DRAGEN TSO500 v2.6.2, BCL Convert in ICA pipeline is parallelized by lane. A node is created to convert BCL to fastq from a particular lane. After demultiplex is complete for all lanes, the fastq files are then aggregated per sample. On average, the turn-around time for BCL Convert per lane is ~35mins.

RNA 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.

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)

• 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:

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.

Variant Filtering Rules

• 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.

Refer to RNA Output for more information.

Downsampling

Each sample is downsampled to 30 million RNA reads. This number represents the total number of single reads (eg, R1 + R2, from all lanes). When using the recommended sequencing configurations or plexity, the samples can have fewer reads than the downsampling limit. In these cases, the FASTQ files are left as-is.

Read Trimming

Reads are trimmed to 76 base pairs for further processing.

RNA Alignment and Fusion Detection

RNA alignment and fusion detection uses trimmed reads in FASTQ format as input. The outputs include a BAM file that contains duplicate-marked read alignments, an SJ.out.tab file that contains unannotated splice junctions, and a CSV file that contains fusion candidates.

DRAGEN aligns RNA reads in a transcript-aware mode using the human hg19 genome containing unplaced contigs (ie, chrUn_gl regions) and uses GENCODEv19 transcript annotations to identify splice sites. DRAGEN identifies and marks duplicate read alignments using start and end coordinates of alignments, which are adjusted for soft clipped reads.

Fusion and splice variant calling only use deduped fragments to score variants. DRAGEN identifies fusion candidates using chimeric split read alignments (pairs of primary and supplementary alignments) against multiple genes. DRAGEN scores and filters reads based on the various features of each candidate such as the number of supporting reads, mapping quality of supporting reads, and sequence homology between parent genes.

The DRAGEN RNA Fusion caller identifies gene fusions by searching for chimeric reads spanning two distinct parent genes. Based on the chimeric reads, DRAGEN first creates a list of fusion candidates, then scores the candidates to report the list of high confidence fusion calls from the candidate pool.

DRAGEN RNA Fusion caller performs the following steps:

1. Generates fusion candidate generation based on split read alignment.

2. Recruits additional evidence from fusion supporting discordant read pairs and soft-clipped reads.

3. Computes fusion candidate features such as gene coverage, read mapping quality, alternate allele frequency, gene homology, alignment anchor length, and breakpoint distance from exon boundary.

4. Scores and ranks the fusion candidates using a logistic regression model.

Note: In v2.6.2 the rna_repeats_hg19.bed file was been updated to include NPM1 to increase sensitivity for that gene (previously this list included only DUX4, SEPT14, CIC, and PSPH).

Splice Variant Calling

RNA splice variant calling is performed for RNA sample libraries. Candidate splice variants (junctions) from RNA Alignment are compared against a database of known transcripts and a splice variant baseline of non-tumor junctions generated from a set of normal FFPE samples from different tissue types. Any splice variants that match the database or baseline are filtered out unless they are in a set of junctions with known oncological function. If there is sufficient read support, the candidate splice variant is kept. This process also identifies candidate RNA fusions.

RNA Fusion Merging

Fusions identified during RNA fusion calling are merged with fusions from proximal genes identified during RNA splice variant calling. These are then annotated with gene symbols or names with respect to a static database of transcripts (GENCODE Release 19). The result of this process is a set of fusion calls that are eligible for reporting

RNA Splice Variant Annotation

The Illumina Annotation Engine annotates detected RNA splice variant calls with transcript-level changes (eg, affected exons in the transcript of a gene) with respect to RefSeq. This RefSeq database is the same RefSeq database used by the small variant annotation process.

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.

HRD Metrics Included in Metrics Output File

Using cell lines or contrived samples for validation or as positive controls may result in inconsistent or unusual results depending on how the samples were constructed. Illumina recommends using commercial controls only for the variant class they are intended for. For example, if the sample is contrived for CNVs but not explicitly for SNV, MSI, TMB, or fusions, we would not recommend using this sample for validation or as a positive control for SNV, MSI, TMB, or fusions.

Demo Data for Commercial Controls

Illumina has provided demo data of some common commercial controls on BaseSpace Sequence Hub on various flow cells:

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)

Refer to for more information.

Downsampling

Each sample is downsampled to 30 million RNA reads. This number represents the total number of single reads (eg, R1 + R2, from all lanes). When using the recommended sequencing configurations or plexity, the samples can have fewer reads than the downsampling limit. In these cases, the FASTQ files are left as-is.

DRAGEN TruSight Oncology 500 Analysis Software can be used to run a subset of samples on different DRAGEN servers to decrease overall processing time. This is possible using a three stage process called scatter/gather, which consists of demultiplexing, analysis, and result gathering.

The first stage is demultiplexing. Demultiplexing runs once on the entire run folder, generates FASTQ files for each sample in the run, and then separates sample files into respective folders. Once complete, note the output directory containing the sample directories holding the FASTQ files.

The process for scattering the analysis on multiple DRAGEN servers is as follows:

1. Determine how many DRAGEN servers are available to run.

2. Run demultiplexing on a single DRAGEN server.

Use the following steps to configure a TruSight™ Oncology 500 run in Illumina Run Manager:

1. Go to the "Runs" section of Illumina Run Manager by selecting "Runs" on the left-hand side

2. Enter sample data manually or by importing a sample sheet

3. To enter sample data run manually, select “Create Run”

Input Validation Steps

In addition to sample sheet validation, before starting an analysis, the software performs the following checks:

• Resource bundle integrity

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.

Prerequisites

The following requirements must be met to install and run DRAGEN TruSight Oncology 500 ctDNA Analysis Application on NovaSeq 6000Dx:

• A NovaSeq 6000Dx sequencing instrument with Illumina Run Manager v1.6.2 installed and paired with DRAGEN Server v4.

The contamination score evaluates presence of sample-to-sample contamination. The algorithm uses common germline SNPs in the homozygous state expected to have variant allele frequencies (VAF) at 0% and 100%. In contaminated samples, the VAFs shift away from the expected values allowing the detection of sample-to-sample contamination.

Overview

A sample sheet is required for each analysis with DRAGEN TruSight Oncology 500 ctDNA Analysis Software. A sample sheet is a comma-separated value (*.csv) file format used by Illumina instruments, platforms, and analysis pipelines to store settings and data for sequencing and analysis. The DRAGEN TruSight Oncology 500 ctDNA Analysis Software is compatible with the sample sheet v2. For general information on the sample sheet v2, refer to .

The sample sheet includes a list of samples and their index sequences, along with additional information required to run DRAGEN TruSight Oncology 500 ctDNA Analysis Software. For example, the library prep kit used for analysis will need to be listed in the sample sheet. Appropriate index adapter sequences are determined by the assay used to perform analysis.

When running analysis on a standalone DRAGEN server or on ICA, a valid sample sheet can be created by:

Auto-launch Prerequisites and Workflow

*The BaseSpace Sequence Hub setting for run monitoring and storage must be selected on the instrument to use DRAGEN TSO 500 analysis auto-launch. For information on preparing your instrument for DRAGEN TSO 500 auto-launch, refer to the documentation for your instrument.

1. Use BaseSpace Sequence Hub Run Planning tool or the sample sheet templates provided on the support page to create and export a sample sheet.

   1. If BaseSpace Run Planning tool is not available in your region, use the sample sheet template.

2. Import the sample sheet to the instrument and start the sequencing run. Refer to for sample sheet guidance.

   1. Data is uploaded to BaseSpace Sequence Hub and then pushed to ICA. You can monitor the run in BaseSpace Sequence Hub.

   2. Analysis auto launches in ICA when sequencing and the upload completes. You can monitor the status of the analysis in BaseSpace Sequence Hub or ICA

   3. If necessary, you can requeue the analysis via BaseSpace Sequence Hub.

3. View the analysis output results in either BaseSpace Sequence Hub or ICA.

BaseSpace Sequence Hub Requirements for ICA Auto-Launch

BaseSpace Run Planning tool is a multi-step workflow that generates a manual launch or auto-launch capable sample sheet for export and requires the following additional settings:

• Access to BaseSpace Sequence Hub.

• ICA Run Storage is enabled under BaseSpace Sequence Hub settings.

Refer to the for information on setting up a BaseSpace Sequence Hub project.

Requeue Analysis

You can requeue analysis of a run via the run's Summary page in BaseSpace Sequence Hub.

Refer to the for more information on requeuing an analysis.

Minimum Storage Requirements on ICA

Refer to the for information on how to manage accounts and subscriptions.

Guided Examples

Please review these guided examples of using DRAGEN TSO 500 Analysis Software with auto-launch on ICA:

Start the DRAGEN TruSight Oncology 500 Analysis Software with the DRAGEN_TSO500-2.6.0.sh Bash script. The script is installed in the /usr/local/bin directory. The Bash script is executed on the command line and runs the software with Docker (or Apptainer if specified).

For arguments, refer to Command-Line Options. You can start from BCL files or from the FASTQ folder produced by BCL Convert. The following requirements apply for both methods:

• Path to the sequencing run or FASTQ folder. Copy the run or FASTQ folder to the DRAGEN server into the staging folder with the following recommended organization: /staging/runs/{RunID}. You can copy the run folder onto the DRAGEN server using Linux commands such as rsync. The sample sheet within the run folder is used unless otherwise specified through the command line.

• Run folder must be intact. Refer to for input requirements.

• If the analysis output folder path is different from the default, provide the analysis output folder path. Refer to .

Storage Requirements

For optimal performance, run analysis on data stored locally on the DRAGEN server. Analysis of data stored on network-attached storage (NAS) can take longer and performance can be less reliable.

The DRAGEN server provides an NVMe SSD in the /staging directory to use as the software output directory. Network-attached storage is required for long-term storage.

When running the DRAGEN TruSight Oncology 500 Analysis Software, use the default settings or set the -analysisFolder command line option to a directory in /staging to make sure the DRAGEN server processes read and write data on the NVMe SSD.

Before beginning analysis, develop a strategy to copy data from the DRAGEN server to a network‑attached storage. Delete output data on the DRAGEN server as soon as possible.

The following are the run and analysis output sizes for each sequencing system per 101 bp:

When launching the analysis, the software checks that the minimum disk space required is available. If the minimum disk space is not available, the software shows an error message and prevents analysis from starting. If disk space is exhausted during a run, the run shows an error and stops analyzing.

The software processes sequencing data to perform quality control, detect variants, determine tumor mutational burden (TMB), microsatellite instability (MSI) status, and genomic instability score (GIS), and report results. The following sections describe the analysis methods used in DRAGEN TruSight Oncology 500 Analysis Software.

DRAGEN TruSight Oncology 500 Analysis Software uses the following workflows to analyze sequencing data.

• FASTQ Generation

• DNA Analysis

  • DNA Alignment and Realignment

  • Read Collapsing

  • Indel Realignment and Read Stitching

  • Small Variant Calling

  • Small Variant Filtering

  • Copy Number Variant (CNV) Calling

  • Phased Variant Calling

  • Variant Merging

  • Annotation

  • Tumor Mutational Burden (TMB) Scoring

  • Microsatellite Instability (MSI) Status

  • Contamination Detection

• RNA Analysis

  • Downsampling

  • Read Trimming

  • Alignment

• Quality Control

  • Run QC

  • DNA Sample QC

  • RNA Sample QC

RNA 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.

DRAGEN TSO 500 analysis software is compatible with several instruments and assay workflows (standard, XP), each of which have implications for the sample sheet.

Sample sheet templates contain all required fields, including index sequences in the proper orientation for all indexes from a given library prep kit. The templates are provided as a starting point for creating a sample sheet manually when launching analysis on a standalone DRAGEN server or on ICA using manual launch.

Users can visit the Sample Sheet guidelines section to learn additional details on required fields and values as they fill-in their sample information. Use the lookup table below to select and download the sample sheet template that matches your instrument, assay, and workflow configuration:

*Lane numbers cannot exceed what is supported by the flow cell in use.

General troubleshooting for a failed sample sheet:

In DRAGEN TruSight Oncology 500 Analysis Software, the analysis fails if a sample sheet is invalid. If an invalid sample sheet in suspected, log files can help troubleshoot a failed analysis. Use the following steps to find the log file for the sample sheet:

1. Navigate to the following location /<analysis_output>/Logs_Intermediates/SamplesheetValidation.

Auto-launch Prerequisites and Workflow

*The BaseSpace Sequence Hub setting for run monitoring and storage must be selected on the instrument to use DRAGEN TSO 500 analysis auto-launch. For information on preparing your instrument for DRAGEN TSO 500 Auto-launch, refer to the documentation for your instrument.

1. Use BaseSpace Sequence Hub Run Planning tool or the sample sheet templates provided on the support page to create and export a sample sheet.

How to Launch Analysis

1. Create a Project: Project can be specific for the DRAGEN TruSight Oncology 500 pipeline or it can contain multiple Pipelines and/or Tools). For information on creating Projects, refer to the Projects section in .

DRAGEN TSO 500 analysis software is compatible with several instruments and assay workflows (standard, XP), each of which have implications for the sample sheet.

Sample sheet templates contain all required fields, including index sequences in the proper orientation for all indexes from a given library prep kit. The templates are provided as a starting point for creating a sample sheet manually when launching analysis on a standalone DRAGEN server or on ICA using manual launch.

Users can visit the section to learn additional details on required fields and values as they fill-in their sample information. Use the lookup table below to select and download the sample sheet template that matches your instrument, assay, and workflow configuration:

Start the DRAGEN TruSight Oncology 500 Analysis Software with the DRAGEN_TSO500.sh Bash script. The script is installed in the /usr/local/bin directory. The Bash script is executed on the command line and runs the software with Docker (or Apptainer if specified).

For arguments, refer to . You can start from BCL files or from the FASTQ folder produced by BCL Convert. The following requirements apply for both methods:

• Path to the sequencing run or FASTQ folder. Copy the run or FASTQ folder to the DRAGEN server into the staging folder with the following recommended organization: /staging/runs/{RunID}. You can copy the run folder onto the DRAGEN server using Linux commands such as rsync. The sample sheet within the run folder is used unless otherwise specified through the command line.

The Illumina DRAGEN TruSight Oncology 500 (TSO 500) ctDNA Analysis Software supports analysis for DNA libraries that are isolated from plasma and prepared using TruSight Oncology 500 ctDNA v2 and v1 assays. The software, which can be run on the DRAGEN Server, on Illumina Connected Analytics, and as an analysis application on NovaSeq 6000Dx, produces a variant call file (VCF) for small variants. Other outputs include tumor mutational burden (TMB), a Jensen-Shannon divergence (sum JSD) score that can be used for evaluating microsatellite instability (MSI) status, files with copy number variants (CNV), fusions as well as coverage reports.

The secondary analysis software starts from a sequencing run folder containing base call files (BCL) or from FASTQ files staged in a FASTQ folder.

This document provides information on installation, configuration, running, troubleshooting as well as analysis algorithms of DRAGEN TruSight Oncology 500 ctDNA analysis software on Illumina Connected Analytics, standalone DRAGEN server, and the NovaSeq 6000Dx analysis application.

The software is optimized for analyzing sequencing outputs generated by the TruSight Oncology 500 ctDNA v2 and v1 assays. Modification might lead to inaccurate data and is a violation of the .

How to Launch Analysis

1. Create a Project: Project can be specific for the DRAGEN TruSight Oncology 500 pipeline or it can contain multiple Pipelines and/or Tools. For information on creating Projects, refer to the Projects section in .

Prerequisites

Illumina Connected Analytics (ICA) subscription includes access to DRAGEN TruSight Oncology 500 Analysis Software. To get started, you need:

• An ICA account with a valid subscription. Refer to the for information on how to register ICA subscription and iCredits.

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.

Scope

This resource provides information on installation, configuration, running, troubleshooting and analysis algorithms for the following software:

• DRAGEN TruSight Oncology 500 Analysis Software v2.6.0

  • for standalone DRAGEN server

  • on Illumina Connected Analytics (ICA)

  • on NovaSeq 6000Dx v2.6.0 (uses a paired DRAGEN server)

• DRAGEN TruSight Oncology 500 Analysis Software on Illumina Connected Analytics (ICA) v2.6.0.8

• DRAGEN TruSight Oncology 500 Analysis Software v2.6.1 (for standalone DRAGEN server)

• DRAGEN TruSight Oncology 500 Analysis Software v2.6.2

  • for standalone DRAGEN server

  • on Illumina Connected Analytics (ICA)

  • on NovaSeq 6000Dx (uses a paired DRAGEN server)

The content is applicable to all software versions unless otherwise specified. The content related to setting up and running the analysis on ICA is only relevant to v2.6.0.

Overview

DRAGEN TruSight™ Oncology 500 Analysis Software supports data analysis for TruSight Oncology 500 Assay , TruSight Oncology 500 High-Throughput Assay, and TruSight Oncology 500 v2 Assay all Research Use Only (RUO).

The software provides local and cloud analysis for DNA and RNA libraries generated from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The assays and the software are optimized to provide high sensitivity and specificity for low-frequency somatic variants across coding exons and additional regions of biological relevance in 523 genes for DNA and RNA biomarkers.

In addition, this software supports data analysis for TruSight Oncology 500 HRD (RUO), which enables detection of homologous recombination deficiency (HRD) through assessment of a genomic instability score (GIS). TSO 500 HRD is available as an optional library prep in TruSight Oncology 500 v2 or as an add-on kit to TruSight Oncology 500.

DNA biomarkers

• Single nucleotide variants (SNVs)

• Insertions

• Deletions

• Copy number variants (CNVs)

DNA Immunotherapy biomarkers

• Tumor mutational burden (TMB)

• Microsatellite instability (MSI)

RNA biomarkers (called from 55 genes)

• Fusions

• Splice variants

Beta features

• Absolute copy numbers (ACN)*

• Loss of heterozygosity (LOH)*

Details of the regions covered by the assays can be found in the assay manifest file. Contact your local Illumina representative for more information.

Local and Cloud Deployments

Local analysis is available using a standalone DRAGEN server or Illumina Run Manager which is a user interface installed on NovaSeq 6000Dx. The software on the standalone DRAGEN server allows for analysis on a single DRAGEN server or splitting across multiple servers.

Cloud analysis is available on Illumina Connected Analytics with auto-launch or manual launch. Both methods are available from BCLs and FASTQs.

Instrument Compatibility

DRAGEN TruSight Oncology 500 analysis software is compatible with data generated on the Illumina instruments as summarized in the table below.

Navigation of Guide

This resource provides information on installation, configuration, running, troubleshooting as well as analysis algorithms of DRAGEN TruSight Oncology 500 analysis software on Illumina Connected Analytics, standalone DRAGEN server, and the NovaSeq 6000Dx analysis application.

Scope

This resource provides information on installation, configuration, running, troubleshooting and analysis algorithms for the following software:

• DRAGEN TruSight Oncology 500 Analysis Software on Illumina Connected Analytics (ICA) v2.5.2

• DRAGEN TruSight Oncology 500 Analysis Software v2.5.3 (for standalone DRAGEN server)

• DRAGEN TruSight Oncology 500 Analysis Software v2.5.4 (for standalone DRAGEN server)

The content is applicable to all 3 software versions unless otherwise specified. The content related to setting up and running the analysis on ICA is only relevant to v2.5.2.

Overview

DRAGEN TruSight™ Oncology 500 Analysis Software supports data analysis for TruSight Oncology 500 Assay and TruSight Oncology 500 High-Throughput Assay, both Research Use Only (RUO).

The software provides local and cloud analysis for DNA and RNA libraries generated from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The assays and the software are optimized to provide high sensitivity and specificity for low-frequency somatic variants across coding exons and additional regions of biological relevance in 523 genes for DNA biomarkers.

In addition, this software supports data analysis for TruSight Oncology 500 HRD (RUO), an optional add-on kit to TruSight Oncology 500, that enables detection of homologous recombination deficiency (HRD) through assessment of a genomic instability score (GIS).

DNA biomarkers:

• Single nucleotide variants (SNVs)

• Insertions

• Deletions

• Copy number variants (CNVs)

DNA Immunotherapy Biomarkers:

• Tumor mutational burden (TMB)

• Microsatellite instability (MSI)

RNA biomarkers (called from 55 genes):

• Fusions

• Splice variants

Beta features:

• Absolute copy numbers (ACN)*

• Loss of heterozygosity (LOH)*

• Tumor fraction*

• Ploidy*

Details of the regions covered by the assays can be found in the assay manifest file. Contact your local Illumina representative for more information.

Local and Cloud Deployments

Local analysis is available using a standalone DRAGEN server. The software on the standalone DRAGEN server allows for analysis on a single DRAGEN server or splitting across multiple servers.

Cloud analysis is available on Illumina Connected Analytics with auto-launch (from BCL) or manual launch (from BCLs and FASTQs), see exceptions for NextSeq 1000/2000 and NovaSeq X in the instrument compatibility tabel below.

Instrument Compatibility

DRAGEN TruSight Oncology 500 analysis software v2.5.x is compatible with data generated on the Illumina instruments as summarized in the table below.

*Among v2.5.x, only DRAGEN TruSight Oncology 500 analysis software v2.5.2 is supported on ICA

**For the data generated with NextSeq 1000/2000 and NovaSeq X, analysis can be started only from FASTQs (not from BCLs). Only manual launch is available for ICA.

[TSO500L_Data] Section

To ensure a successful analysis, follow these guidelines:

1. Avoid any blank lines at the end of the sample sheet; these can cause the analysis to fail.

2. When running local analysis using the command line save the sample sheet in the sequencing run folder with the default name SampleSheet.csv, or choose a different name and specify the path in the command-line options.

ICA with Auto-launch: Sample Sheet Requirements

Refer to the following requirements to create sample sheets for running the analysis on ICA with Auto-launch. For sample sheet requirements common between deployments see . Samples sheets can be created using BaseSpace Run Planning Tool or manually by downloading and editing a sample sheet template

[Cloud_TSO500L_Data] Section

Refer to for this section's requirements.

[Cloud_TSO500L_Settings] Section

[Cloud_Data] Section

[Cloud_Settings] Section

NovaSeq 6000Dx Analysis Application: Sample Sheet Requirements

This section describes fields specific for sample sheets for NovaSeq 6000Dx Analysis Application. For more information on DRAGEN TSO 500 ctDNA Analysis Software sample sheet requirements, refer to the sections above.

[BCLConvert_Settings] Section

Introduction

The NovaSeq X platform supports loading samples from different assays into different lanes within a single sequencing run. To ensure compatibility, the DRAGEN TSO 500 analysis software (v2.6.0 and above) supports sample sheets containing multiple data sections, with one section per assay (for example, TSO500S_Data and TSO500L_Data).

This section describes how the software validates and processes multi-assay sample sheets and details the specific rules and logic applied during validation.

Planning a Run with a Multi-Assay Flow Cell

1. In the BaseSpace Sequence Hub home page, click "Runs" -> "New Run" -> "Run Planning".

2. In the "Create a Run" page, select "NovaSeq X Series" as instrument platform.

3. Provide information for the first assay until the "Run Review" page.

   • For TSO 500 or TSO 500 ctDNA assays, to auto-launch multiple pipelines at once, set "" to True

Analyzing Data from a Multi-Assay Flow Cell

Sample Sheet Validation

The sample sheet validator determines which data section(s) to validate based on the active workflow type. The list of samples to process is generated only from the relevant section (for example, Solid or ctDNA). This ensures that only valid samples for the selected workflow are validated.

The workflow’s data section may contain fewer samples than the BCLConvert_Data section. However, all samples listed in the workflow data section must exist in BCLConvert_Data section. The downstream pipeline will only process samples found in the workflow-specific data section.

Library Prep Kits

Multi-assay sample sheets may include multiple library prep kits values separated by semicolons (";").

Validation Logic:

Adapter Reads

When working with multiple assays, the "AdapterRead" field in the sample sheet may include multiple adapter sequences separated by "+". The adapter read of the selected workflow will be filled in the intermediate sample sheet.

Override Cycles

Override cycles are determined by the index and read length specified in the "Reads" section of the RunInfo.xml file and assay specifics. For multi-assay flow cells, the index and read length provided in RunInfo.xml should be the longer of the sequencing cycles required by the two assays. Override cycles for assays requiring shorter indexes and read lengths will be padded to match the cycle lengths used in the run.

BCL Convert Settings and Data

Starting from BCL:

• Sample Sheet Validator recalculates and inserts the correct override cycles into the BCLConvert_Data section of the intermediate sample sheet.

  • If index lengths vary, the logic has been enhanced to automatically pad shorter indexes with "N" to standardize cycle lengths.

• Sample Sheet Validator also writes in values for: Adapter Reads, MaskShortReads, AdapterBehavior, MinimumTrimmedReadLength. These values are based on the TSO 500 library prep kit determined from the input sample sheet.

Starting from FASTQ:

• If users run BCL Convert separately to generate FASTQ files:

  • The input sample sheet must already contain valid override cycles, adapter reads, and other BCL Convert Settings.

  • The resulting TSO 500 workflow run will start from FASTQ input files.

  • BCLConvert_Settings and BCLConvert_Data sections will be excluded from the intermediate sample sheet created by the TSO 500 workflow.

BaseSpace Autolaunch

Analyses of samples in multi-assay flow cells can be launched simultaneously from BaseSpace. As long as "Starts from FASTQ" is set to True in Analysis Settings, all samples from a multi-assay flow cell will be demultiplexed first to generate FASTQ files. Then, the ICA pipelines (e.g. DRAGEN TSO 500, DRAGEN TSO 500 ctDNA) will be launched simultaneously to process the samples for each of the assays.

Summary of Rule Changes

You can use the following command-line options with DRAGEN TruSight Oncology 500 Analysis Software.

To learn more about the input requirements, use the --help command-line option.

Overview

The installation script for DRAGEN TruSight Oncology 500 Analysis Software installs the following software and dependencies:

1. DRAGEN TruSight Oncology 500 Analysis Software itself

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.

Introduction

The NovaSeq X platform supports loading samples from different assays into different lanes within a single sequencing run. To ensure compatibility, the DRAGEN TSO 500 ctDNA v2.6.3 analysis software supports sample sheets containing multiple data sections, with one section per assay (for example, TSO500S_Data and TSO500L_Data).

This section describes how the software validates and processes multi-assay sample sheets and details the specific rules and logic applied during validation.