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 TruSight Oncology 500 Assay and TruSight Oncology 500 High-Throughput Assay, both Research Use Only (RUO)

• DRAGEN TruSight™ Oncology 500 ctDNA Analysis Software supports TruSight Oncology 500 ctDNA assay v1 and v2, both Research Use Only (RUO)

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.

Here are the articles in this section:

Prerequisites

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

• Illumina Run Manager installed by Illumina support personnel

• TSOCombined and TSO500_HRD licenses installed by Illumina support personnel

  • TSOCombined 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 at customercare@illumina.com to obtain installation package for Illumina DRAGEN TruSight Oncology 500 (HRD) Analysis Application on NovaSeq 6000Dx

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

1. The installation package contains the following:

   1. DRAGEN TSO 500 Analysis Application: DRAGEN_TSO500HRD_v2.6.0-2v12.iapp

   2. DRAGEN ires: drageninstaller_3.10.17-8.el8.x86_64_prod.ires

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

   4. The installation is complete once the DRAGEN version 3.10.17 is in the install version(s) list

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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

2. DRAGEN Software if a compatible version is not present

3. Docker software if a compatible version is not present

4. A script required to generate DRAGEN genome hash table

5. A script to check that DRAGEN TruSight Oncology 500 Analysis Software is installed properly

Installation Requirements

Hardware

• DRAGEN server v3 or v4

• Network-attached storage (NAS) with enabled mkfifo if performing analysis for the TruSight Oncology 500 High-Throughput assay

Software

• Linux CentOS 7.9 operating system (or later) or Oracle Linux 8 (or later), one of which is provided on the server. Oracle Linux 8 is recommended.

• Docker Software, see table below for minimum version needed. If sufficient Docker software is not present on the server, the TSO 500 installer will install compatible Docker software.

• DRAGEN Server Software*, see table below for minimum version needed as the host version on the server. If sufficient DRAGEN software is not present on the server, the TSO 500 installer will install compatible DRAGEN software.

Licenses

• TSOCombined license

• TSO500_HRD license (to analyze data generated with the TSO 500 HRD add-on kit)

Permissions

Illumina recommends logging in as root user for installation, but as a non-root user for running TSO 500 analysis.

• Installing and uninstalling DRAGEN TruSight Oncology 500 Analysis Software and running the system check requires root privileges.

• Run DRAGEN TruSight Oncology 500 Analysis Software without being logged in as a root user. Running the DRAGEN TruSight Oncology 500 Analysis Software as root is not required or recommended.

Compatibility with other DRAGEN pipelines

Software versions without multi-version compatibility referred to as single-version compatible. DRAGEN TSO 500 Analysis Software v2.6.1 will disrupt installations of single-version compatible software from the DRAGEN server. To uninstall a previous version of DRAGEN TSO 500 Analysis Software, refer to the respective guide.

Compatibility of software for co-installation with DRAGEN TSO 500 v2.6.1 on a DRAGEN server is summarized in the table below:

*DRAGEN TSO 500 Analysis Software v2.6.1 can run on a single server with another multi-version compatible DRAGEN TSO 500 Analysis Software, e.g. DRAGEN TSO 500 v2.5.4 (v2.5.4 should be installed before installing v2.6.1). DRAGEN TSO 500 Analysis Software v2.6.1 can be co-installed with multi-version compatible DRAGEN TSO 500 ctDNA Analysis Software or other DRAGEN pipelines with any order of installation.

**For example, DRAGEN Enrichment, DRAGEN Germline, DRAGEN WGS Heme v1.0.0 and others. Order of installation does not matter.

Installation Instructions

As a root user, perform the following steps to install DRAGEN TruSight Oncology 500 v2.6.1 Analysis Software:

2. Download the installation package provided in the email from Illumina. The link expires after 7 days.

1. Make sure no other analysis is being performed. Installing the software while performing other analyses prevent the installer process from proceeding

2. Copy the install script to the /staging directory to store the script in the directory.

1. Use the following command to update the run script permission: chmod +x /staging/install_DRAGEN_TSO500-2.6.1.run

2. Use the following command to run the installation script, which runs for approximately 20 minutes:

   1. For Docker, use the following command: sudo TMPDIR=/staging /staging/install_DRAGEN_TSO500-2.6.1.run . The script installs compatible DRAGEN software and removes any previously installed versions.

   2. For Apptainer, use the following command: sudo TMPDIR=/staging /staging/install_DRAGEN_TSO500-2.6.1.run -- --noDockerInstall This will not install Apptainer, but will install the analysis software in the SIF container format and modify the software to launch analyses using Apptainer.

3. During the installation process, you might be instructed to reboot or power cycle the system to complete the installation of the DRAGEN software. A power cycle of the system requires the server be shut down and restarted.

4. Log out of the server and then log back in.

License Installation

To install a license (TSOCombined and/or TSO500_HRD) on a DRAGEN server connected to the internet:

1. Confirm that the server is connected to the Internet, example: ping www.illumina.com

2. Run the following command: /usr/bin/dragen_lic -i auto

To install a license (TSOCombined and/or TSO500_HRD) on a DRAGEN server not connected to the internet:

2. Download and save the license file(s) to a location that is accessible from the DRAGEN server

3. For each license file, run the command, where <license file received> is the absolute path to the license file: sudo /usr/bin/dragen_lic -i /tmp/<license file received>.bin

To check the success of license installation, run: /usr/bin/dragen_lic. Installed licenses should be in the list.

Running the System Check

After installation is complete, make sure the system functions properly by running the following command: /usr/local/bin/check_DRAGEN_TSO500-2.6.1.sh

The script checks that:

• All required services are running

• Proper Docker image is installed

• DRAGEN TruSight Oncology 500 Analysis Software can successfully process a test data set

The system check script runs for approximately 25 minutes. If the script prints a failure message, contact Illumina Technical Support and provide the /staging/check_DRAGEN_TSO500_<timestamp>.tgz output file.

If using MacOS to connect to a server, an error can occur if the local settings are not in English. To resolve the error, disable the ability to set environment variables automatically in Terminal settings.

Uninstall Software

The DRAGEN TruSight Oncology 500 Analysis Software installation includes an uninstall script called uninstall_DRAGEN_TSO500-2.6.1.sh, which is located in /usr/local/bin.

Executing the uninstall script removes the following assets:

• All DRAGEN TruSight Oncology 500 Analysis Software related scripts located in /usr/local/bin

• Resources found in /staging/illumina/DRAGEN_TSO500

• The dragen_tso500:2.6.1: Docker image

To uninstall the DRAGEN TruSight Oncology 500 Analysis Software, run the following command as a root user:

uninstall_DRAGEN_TSO500-2.6.1.sh

You are not required to uninstall Docker or DRAGEN software. To remove Docker, review the install instructions for your operating system in the Docker documentation.

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

• A positive balance of iCredits for data storage

Overview

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 running analysis using a NovaSeq 6000Dx Analysis Application, a valid sample sheet can be created by:

The run set up section of this guide includes specific instructions to plan a run and set up a valid sample sheet for each deployment of DRAGEN TruSight Oncology 500 Analysis Software.

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”

4. Choose "DRAGEN TruSight™ Oncology 500 (with HRD) Analysis Application" from the "Create Run" screen to set-up and analyze runs for TruSight Oncology 500 assay with or without HRD add-on

Run Settings

1. On the "Run Settings" screen, enter a run name with the following criteria:

   1. 1 - 40 characters.

   2. Alphanumeric characters, underscores, or dashes only.

   3. Unique across all runs on the instrument.

   The run name identifies the run from sequencing through analysis.

2. [Optional] Enter a run description. The run description must have the following criteria:

   1. 1 - 50 characters.

   2. Alphanumeric characters or spaces only.

   3. Spaces must be preceded and followed by an alphanumeric character.

3. Select kit used during library preparation:

   1. TruSight Oncology 500

   2. TruSight Oncology 500 High-Throughput

4. Index adapter kit will be automatically selected based on the library prep kit selection

5. [Optional] Enter a library tube ID.

Depending on the library prep kit selected, additional fields will be populated for run settings and are not editable. Read and index lengths will differ between library prep kit type.

Sample Data

Use the table on the "Sample Data" screen to enter sample information manually.

Alternately, select Import Samples to upload sample information. Refer to NovaSeq 6000Dx Analysis Application: Sample Sheet Requirements for sample sheet requirements.

1. Select lane information. Options include one to four, or all lanes.

2. Enter a unique sample ID in the sample ID field with the following criteria:

   1. Controls should be added first.

   2. 1 - 40 characters.

   3. Alphanumeric characters, underscores, or dashes only.

   4. Underscores and dashes must be preceded and followed by an alphanumeric character.

3. Select an index set ID for the DNA / RNA library prepared from the sample.

4. [Optional] Enter a library name.

Depending on the options selected for index set ID, additional fields will be auto-populated for sample data and are not editable.

Sample Settings

Use the table on the "Sample Settings" screen to enter additional sample information.

1. Enter Pair ID with the following criteria:

   1. 1 - 40 characters.

   2. Alphanumeric characters, underscores, or dashes only.

   3. Underscores and dashes must be preceded and followed by an alphanumeric character.

   4. Pairs at most one DNA and one RNA samples from the same biological sample from the same individual.

2. Select Sample Type: DNA or RNA

3. Enter Sample Feature: Select HRD for DNA samples with HRD probes. For all other samples, leave the field blank.

4. [Optional] Enter a sample name with the following criteria:

   1. 1 - 50 characters.

   2. Alphanumeric characters, dashes, underscores, or spaces.

   3. Spaces, underscores, and dashes must be preceded and followed by an alphanumeric character.

5. [Optional] Enter a sample description with the following criteria:

   1. 1 - 50 characters.

   2. Alphanumeric characters, dashes, underscores, or spaces.

   3. Spaces, underscores, and dashes must be preceded and followed by an alphanumeric character.

Additional fields will be auto-populated based on selections made in the Sample Data screen, which are not editable.

Before starting your run, review that the information entered is correct in the “Run Review” page before saving.

Standard Sample Sheet Requirements

The following sample sheet requirements describe required and optional fields for DRAGEN TSO 500 Analysis Software. Depending on the deployment (standalone DRAGEN server, ICA with auto-launch, ICA with manual launch, NovaSeq 6000Dx analysis application), certain sections and required values can deviate from the standard requirements. These deviations are noted in the information below.

Use the following steps to create a valid sample sheet.

1. Download the sample sheet v2 template that matches the instrument & assay run.

2. In the Sequencing Settings section, enter the following required parameters:

[Sequencing_Settings] Section

1. In the BCL Convert Settings section, enter the following required parameters:

[BCLConvert_Settings] Section

1. In the BCL Convert Data section, enter the following parameters for each sample.

[BCLConvert_Data] Section

1. In the TSO 500 Data section, enter the following parameters:

[TSO500S_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 Standard Sample Sheet Requirements. Samples sheets can be created using BaseSpace Run Planning Tool or manually by downloading and editing a sample sheet template

[Cloud_TSO500S_Data] Section

Refer to [TSO500_Data] Section for this section's requirements.

[Cloud_TSO500S_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 Analysis Software sample sheet requirements, refer to the sections above.

[BCLConvert_Settings] Section

[TSO500HRD_Data] Section

Refer to [TSO500S_Data] Section for this section's requirements.

How to Create TSO 500 Sample Sheets in BaseSpace Run Planning tool

The BaseSpace Sequence Hub Run Planning tool is available, and is used to generate a valid sample sheet in v2 format for use on a TSO 500 supported sequencer for both ICA and Standalone DRAGEN Server analysis options. Filling out the form on the user interface will produce a exportable sample sheet with the required fields filled in. Refer to ICA Auto-launch Sample Sheet Requirements for descriptions of fields that appear in ICA sample sheets.

The sections below represent each step in the BaseSpace Run Planning tool.

Step 1: Run Settings

Step 2: Configuration

Step 3: Sample Settings

Users can manually enter sample information, or download a template file to bulk upload sample information. Users can import the completed template or a compatible sample sheet.

Step 4: Run Review

Once all details are captured and pass validation, the user can review the details on the Run Review screen. From here they can choose to edit details in previous screens or export the sample sheet. Once completed, press the Cancel button to finish run planning.

Note: once leaving this screen, the run and sample sheet will not be accessible.

For NovaSeqX Plus users, the run can be saved as a draft or as a planned run (via “Save as Draft” and “Save as Planned” buttons respectively). Either selection will save the run to the Planned Runs screen on BaseSpace. There is no option to export the sample sheet on this screen.

Planned Runs Screen (NovaSeq X Series only)

The Planned Runs screen lists all planned or drafted runs. Users can set drafted runs to planned, export the sample sheet, and edit or delete a run on this screen.

Once the run is saved as Planned, it will appear on the NovaSeq X Series instrument where it can be selected for sequencing.

For more information on run planning, refer to the BaseSpace Sequence Hub support site page.

Guided Examples

Please review these guided examples of analysis workflows that include a step of setting up a run in BaseSpace Run Planning tool:

• NovaSeq 6000Dx: TSO 500 Auto-launch Analysis in Cloud

• NextSeq 500/550Dx: TSO 500 and Connected Insights Auto-launch Analysis in Cloud

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.

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)

• DRAGEN TruSight Oncology 500 Analysis Software on Illumina Connected Analytics (ICA) v2.6.0

• DRAGEN TruSight Oncology 500 Analysis Application on NovaSeq 6000Dx v2.6.0 (uses a paired DRAGEN server)

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

• Exon-level CNVs

• Multinucleotide variants (MNVs)

• Genomic Instability Score (GIS Score) *

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 or an application with a user interface 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.

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 Starting from BCL Files for input requirements.

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

Storage Requirements

For optimal performance, run analysis on data stored locally on the DRAGEN server. Analysis of data stored on 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.

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 Node Analysis Output Folder Structure

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.

  • 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, exon coverage report bed, 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, exon coverage report bed 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.

• Results

  • Metrics Output TSV (all pair IDs)

  • Pair 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 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

      • Exon Coverage Report TSV

      • Gene Coverage Report TSV

Multiple Node Analysis Output Folder Structure

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:

  • 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.

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 pair ID.

• Results

  • MetricsOutput.tsv

  • Sample_1

  • Sample_2

  • Sample_<#>

  • _tags.json

The Results subfolder contains the following files:

• Results

  • MetricsOutput.tsv

  • <Pair_id>

    • CombinedVariantOutput.tsv

    • <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

Logs_intermediates Folder Structure

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

  • CombinedVariantOutput

  • FastqGeneration

  • MetricsOutput

  • DnaDragenExonCnvCaller

  • DnaFastqValidation

  • DNACoverageReport

  • Gis

  • Tmb

  • SampleAnalysisResults

  • SampleSheetValidation

  • passing_sample_steps.json

  • RnaFusion

  • Contamination

  • Annotation

  • RnaAnnotation

  • RnaDragenCaller

  • RnaSpliceVariantCalling

  • RunQc

  • FastqDownsample

  • PassingSampleSteps

  • ResourceVerification

  • LrCalculator

  • LrAnnotation

  • RnaQcMetrics

  • RnaFastqValidation

  • RNACoverageReport

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

• 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

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

2. DRAGEN Software if a compatible version is not present

3. Docker software if a compatible version is not present

4. A script required to generate DRAGEN genome hash table

5. A script to check that DRAGEN TruSight Oncology 500 Analysis Software is installed properly

Installation Requirements

Hardware

• DRAGEN server v3 or v4

• If performing analysis for the TruSight Oncology 500 High-Throughput assay, mkfifo needs to be enabled on the network-attached storage (NAS).

Software

• By default Linux CentOS 7.9 operating system (or later) or Oracle Linux 8 (or later), is provided. Oracle Linux 8 is recommended.

• Docker Software, see table below

• DRAGEN Software, see table below

Licenses

• TSOCombined license

• TSO500_HRD license (to analyze data generated with the TSO 500 HRD add-on kit)

Permissions

Illumina recommends logging in as root user for installation, but as a non-root user for running TSO 500 analysis.

• Installing and uninstalling DRAGEN TruSight Oncology 500 Analysis Software and running the system check requires root privileges.

• Run DRAGEN TruSight Oncology 500 Analysis Software without being logged in as a root user. Running the DRAGEN TruSight Oncology 500 Analysis Software as root is not required or recommended.

Compatibility with other TruSight Oncology 500 and TruSight Oncology 500 ctDNA Analysis Software

DRAGEN TruSight Oncology 500 Analysis Software v2.6.0 can be installed on one DRAGEN server with:

1. DRAGEN TruSight Oncology 500 ctDNA Analysis Software v2.6.0 (v3.10.17*)

2. One prior 2.x version of DRAGEN TruSight Oncology 500 ctDNA Analysis Software (v2.1.1 (v3.10.9*), v2.5.0 (v3.10.15*), 2.6.0 (v3.10.17*), 2.6.1 (v3.10.18*))

3. One prior 2.x version of DRAGEN TruSight Oncology 500 Analysis Software (v2.1.1 (v3.10.9*), v2.5.3 (v3.10.16*)

*DRAGEN Software version

Contrary to the prior versions, the installation scripts for DRAGEN TruSight Oncology 500 Analysis Software v2.6.0 and DRAGEN TruSight Oncology 500 ctDNA v2.6.0 do not uninstall previous versions of DRAGEN TruSight Oncology 500 Analysis Software. To uninstall a previous version of DRAGEN TruSight Oncology 500 Analysis Software, refer to the respective guide.

Installation Instructions

As a root user, perform the following steps to install DRAGEN TruSight Oncology 500 v2.6.0 Analysis Software:

2. Download the installation package provided in the email from Illumina. The link expires after 7 days.

1. Make sure no other analysis is being performed. Installing the software while performing other analyses prevent the installer process from proceeding

2. Copy the install script to the /staging directory to store the script in the directory.

1. Use the following command to update the run script permission: chmod +x /staging/install_DRAGEN_TSO500-2.6.0.run

2. Use the following command to run the installation script, which runs for approximately 20 minutes:

   1. For Docker, use the following command: sudo TMPDIR=/staging /staging/install_DRAGEN_TSO500-2.6.0.run . The script installs compatible DRAGEN software and removes any previously installed versions.

   2. For Apptainer, use the following command: sudo TMPDIR=/staging /staging/install_DRAGEN_TSO500-2.6.0.run -- --noDockerInstall This will not install Apptainer, but will install the analysis software in the SIF container format and modify the software to launch analyses using Apptainer.

3. During the installation process, you might be instructed to reboot or power cycle the system to complete the installation of the DRAGEN software. A power cycle of the system requires the server be shut down and restarted.

4. Log out of the server and then log back in.

License Installation

To install a license (TSOCombined and/or TSO500_HRD) on a DRAGEN server connected to the internet:

1. Confirm that the server is connected to the Internet, example: ping www.illumina.com

2. Run the following command: /opt/edico/bin/dragen_lic -i auto

To install a license (TSOCombined and/or TSO500_HRD) on a DRAGEN server not connected to the internet:

2. Download and save the license file(s) to a location that is accessible from the DRAGEN server

3. For each license file, run the command, where <license file received> is the absolute path to the license file: sudo /opt/edico/bin/dragen_lic -i /tmp/<license file received>.bin

To check the success of license installation, run: /opt/edico/bin/dragen_lic. Installed licenses should be in the list.

Running the System Check

After installation is complete, make sure the system functions properly by running the following command: /usr/local/bin/check_DRAGEN_TSO500-2.6.0.sh

The script checks that:

• All required services are running

• Proper Docker image is installed

• DRAGEN TruSight Oncology 500 Analysis Software can successfully process a test data set

The system check script runs for approximately 25 minutes. If the script prints a failure message, contact Illumina Technical Support and provide the /staging/check_DRAGEN_TSO500_<timestamp>.tgz output file.

If using MacOS to connect to a server, an error can occur if the local settings are not in English. To resolve the error, disable the ability to set environment variables automatically in Terminal settings.

Uninstall Software

The DRAGEN TruSight Oncology 500 Analysis Software installation includes an uninstall script called uninstall_DRAGEN_TSO500-2.6.0.sh, which is located in /usr/local/bin.

Executing the uninstall script removes the following assets:

• All DRAGEN TruSight Oncology 500 Analysis Software related scripts located in /usr/local/bin

• Resources found in /staging/illumina/DRAGEN_TSO500

• The dragen_tso500:2.6.0: Docker image

To uninstall the DRAGEN TruSight Oncology 500 Analysis Software, run the following command as a root user:

uninstall_DRAGEN_TSO500-2.6.0.sh

You are not required to uninstall Docker or DRAGEN software. To remove Docker, review the install instructions for your operating system in the Docker documentation.

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.

1. Transfer the FASTQ folder output from the original DRAGEN server to additional servers.

   1. Logs_Intermediates/FastqGeneration.

2. Run analysis software using the --fastqFolder option on both the original and additional DRAGEN servers.

   • Option 1 Copy the original SampleSheet.csv to each server. Then provide a subsetted list to the Bash script on each DRAGEN server with the intended samples/pairs to run.

   • Option 2 Copy and modify the SampleSheet.csv to each DRAGEN server to only contain the list of samples/pairs to run. The software verifies that all samples in the sample sheet are contained within the FASTQ folders unless the --sampleOrPairIDs command-line option is present in the analysis launch. Failure to account for these checks results in an error.

3. Copy the results from demultiplexing and each analysis run onto a single server, and then generate the final /Results directory, which contains the aggregated results. Enter the --gather command followed by the output directories of the demultiplexing step and each individual analysis run.

Commands for Multinode Analysis

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. 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.

Requeue Analysis

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

Minimum Storage Requirements on ICA

Guided Examples

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

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.

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.

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.

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.

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.

Note:

• Use full paths when specifying the file paths in the command line.

• Avoid special characters such as &, *, #, and spaces.

• When starting from BCL files, only the run folder needs to be specified. The immediate parent directory containing the BCL files does not need to be specified.

When running the analysis software using SSH, Illumina recommends using additional software to prevent unexpected termination of analysis. Illumina recommends screen and tmux.

1. Wait for any running DRAGEN TruSight Oncology 500 Analysis Software containers to complete before launching a new analysis. Run the following command to generate a list of running containers:docker ps

2. Select from one of the following options:

• Start from BCL files in the run folder with the sample sheet included in the run folder. DRAGEN_TSO500-2.6.0.sh \ --runFolder /staging/{RunFolderName} \ --analysisFolder /staging/{AnalysisFolderName}

• Start from BCL files in the run folder with the sample sheet located in a folder other than the run folder. DRAGEN_TSO500.sh \ --runFolder /staging/{RunFolderName} \ --analysisFolder /staging/{AnalysisFolderName} \ --sampleSheet /staging/{SampleSheetName}.csv

• Start from BCL files in the run folder with a different sample sheet and demultiplexing only. DRAGEN_TSO500-2.6.0.sh \ --runFolder /staging/{RunFolderName} \ --analysisFolder /staging/{AnalysisFolderName} \ --sampleSheet /staging/{SampleSheetName}.csv \ --demultiplexOnly

• Start from FASTQ with the sample sheet included in the FASTQ folder and with different resources and hash table folders. DRAGEN_TSO500-2.6.0.sh \ --resourcesFolder /staging/illumina/DRAGEN_TSO500/resources \ --hashtableFolder /staging/illumina/DRAGEN_TSO500/ref_hashtable \ --fastqFolder /staging/{FastqFolderName} \ --analysisFolder /staging/{AnalysisFolderName}

• Start from FASTQ folder with sample sheet included in the FASTQ folder and subset of samples or pairs. DRAGEN_TSO500-2.6.0.sh \ --fastqFolder /staging/{FastqFolderName} \ --analysisFolder /staging/{AnalysisFolderName} \ --sampleOrPairIDs "Pair_1,Pair2"

Starting from BCL Files

If starting from BCL (*.bcl) files, DRAGEN TruSight Oncology 500 Analysis Software requires the run folder to contain certain files and folders. These inputs are required for Docker.

The run folder contains data from the sequencing run, make sure that the folder contains the following files:

Starting from FASTQ Files

The following inputs are required for running the DRAGEN TruSight Oncology 500 Analysis Software using FASTQ (*.fastq) files. The requirements apply to Docker.

• Full path to an existing FASTQ folder.

• The sample sheet is in the FASTQ folder path, or you can set the path to the sample sheet with the --sampleSheet override command line option.

Make sure there is sufficient disk space for the analysis to complete. Refer to the --help command line argument details for disk space requirements.

FASTQ File Organization

Store FASTQ files in individual subfolders that correspond to a specific Sample_ID. Keep file pairs together in the same folder. Alternatively, store the FASTQ files in one flat folder structure where the FASTQ files are stored in one folder.

The DRAGEN TruSight Oncology 500 Analysis Software requires separate FASTQ files per sample. Do not merge FASTQ files.

The instrument generates two FASTQ files per flow cell lane, so that there are eight FASTQ files per sample.

Sample1_S1_L001_R1_001.fastq.gz

• Sample1 represents the Sample ID.

• The S in S1 means sample, and the 1 in S1 is based on the order of samples in the sample sheet, so S1 is the first sample.

• L001 represents the flow cell lane number.

• The R in R1 means Read, so R1 refers to Read 1.

Methods for Launching Analysis

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

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

Refer to RNA Analysis Methods for more information.

Splice Variant VCF

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.

Splice Variant Annotated JSON

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

• Canonical

• Consequence

All Fusions CSV

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