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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:
Instructions to install DRAGEN TSO 500 Analysis Application on NovaSeq 6000Dx (RUO mode).
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
Contact Illumina Customer Care at customercare@illumina.com to obtain installation package for Illumina DRAGEN TruSight Oncology 500 (HRD) Analysis Application on NovaSeq 6000Dx
Download the installation package provided in the email by Illumina Customer Care. The link will expire after 7 days.
It is recommended to use a command line tool like wget or curl to download the file rather than pasting the link into the web browser bar. For example:
curl -o {filename} "{link}"
wget -O {filename} '{link}'
Where the file name is the name of either DRAGEN TSO 500 Analysis Application file or DRAGEN ires file, and the link is provided by Illumina Customer Care.
The installation package contains the following:
DRAGEN TSO 500 Analysis Application: DRAGEN_TSO500HRD_v2.6.0-2v12.iapp
DRAGEN ires: drageninstaller_3.10.17-8.el8.x86_64_prod.ires
MD5sum for the installation package contents are as follows:
drageninstaller_3.10.17-8.el8.x86_64_prod.ires
12df06502776d8b673c73fb714dc466a
DRAGEN_TSO500HRD_v2.6.0-2v12.iapp
dc4fe5fe2fc3eca57969e978886dcedf
Install the DRAGEN version using the ires:
Log into Illumina Run Manager as a user with admin credentials
Navigate to the top left menu to "DRAGEN" in the drop down
Select "Add DRAGEN Installer" and upload the DRAGEN ires file
The installation is complete once the DRAGEN version 3.10.17 is in the install version(s) list
\
The installation script for DRAGEN TruSight Oncology 500 Analysis Software installs the following software and dependencies:
DRAGEN TruSight Oncology 500 Analysis Software itself
DRAGEN Software if a compatible version is not present
Docker software if a compatible version is not present
A script required to generate DRAGEN genome hash table
A script to check that DRAGEN TruSight Oncology 500 Analysis Software is installed properly
DRAGEN server v3 or v4
Network-attached storage (NAS) with enabled mkfifo if performing analysis for the TruSight Oncology 500 High-Throughput assay
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.
*The DRAGEN Server Software version may be higher than the DRAGEN version used by the DRAGEN TSO 500 v2.6.1 pipeline (DRAGEN v3.10.17), which is provided inside the DRAGEN TSO 500 docker image.
TSOCombined
license
TSO500_HRD
license (to analyze data generated with the TSO 500 HRD add-on kit)
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.
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.
As a root user, perform the following steps to install DRAGEN TruSight Oncology 500 v2.6.1 Analysis Software:
Download the installation package provided in the email from Illumina. The link expires after 7 days.
It is recommended to use a command line tool like wget or curl to download the file rather It is recommended to use a command line tool like wget or curl to download the file rather than pasting the link into the web browser bar. For example:
curl -o {filename} "{link}"
wget -O {filename} '{link}'
Where the file name is the installation script file name, and the link is provided by Illumina Customer Care.
Make sure no other analysis is being performed. Installing the software while performing other analyses prevent the installer process from proceeding
Copy the install script to the /staging
directory to store the script in the directory.
Installation Script: install_DRAGEN_TSO500-2.6.1.run
MD5sum value: sha256:00326573934d73e16b8b9d0f7790b58da5ac8112875deb359d2944f95b95aed5
Use the following command to update the run script permission:
chmod +x /staging/install_DRAGEN_TSO500-2.6.1.run
Use the following command to run the installation script, which runs for approximately 20 minutes:
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.
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.
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.
Log out of the server and then log back in.
To install a license (TSOCombined
and/or TSO500_HRD
) on a DRAGEN server connected to the internet:
Confirm that the server is connected to the Internet, example: ping www.illumina.com
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:
Download and save the license file(s) to a location that is accessible from the DRAGEN server
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.
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.
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.
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.
TSOCombined
license has been pre-installed to DRAGEN servers in manufacturing since August 2022 and TSO500_HRD
since February 2025 and additionally distributed to DRAGEN servers connected online. To generate a list of installed DRAGEN server licenses, run the following command: /usr/bin/dragen_lic
. If a license is not installed, contact Illumina Customer Care at for the license.
A non-root user must be a member of the Docker group to run Docker. For more information on Docker permission requirements and alternatives to running as root, refer to the Docker documentation available on the .
DRAGEN TSO 500 Analysis Software v2.6.1 is multi-version compatible. Multi-version compatibility refers to ability to be installed on a single DRAGEN server with software running a different version of DRAGEN software. For example, multi-version compatible pipelines running DRAGEN v4.3.6 can be co-installed on a server alongside DRAGEN TSO 500 pipelines running DRAGEN v3.10.17. For more details on DRAGEN multi-version compatibility, please visit .
Contact Illumina Customer Care at to obtain the DRAGEN TruSight Oncology 500 Analysis Software installer package.
Use the following command to build the DRAGEN server hash table, which runs for approximately 60 minutes:
/usr/local/bin/build-hashtable_DRAGEN_TSO500-2.6.1.sh
Refer to if any errors occur.
Review license requirements, how to check which licenses are installed and how to receive a license in . Licenses can be installed before or after DRAGEN TSO 500 software installation.
Contact Customer Care at to request a license file for each of the needed licenses
Refer to the for information on how to register ICA subscription and iCredits.
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 .
BaseSpace Run Planner (preferred), see for details
Downloading and modifying a sample sheet template following the requirements, see for details
Using the user interface of the DRAGEN TruSight Oncology 500 Analysis Application, see for details
Downloading and modifying a sample sheet template following the requirements (see for details), then importing it to Illumina Run Manager.
Docker
20.10 or greater
Docker 20.10.15
DRAGEN Server Software*
v3.10.x, where x >=19, v4.3+
DRAGEN Software 3.10.19
DRAGEN TSO 500
2.6.0
Single-version
No
DRAGEN TSO 500
2.5.4
Multi-version
Yes*
DRAGEN TSO 500
2.5.3 or below
Single-version
No
DRAGEN TSO 500 ctDNA
2.6.2+
Multi-version
Yes
DRAGEN TSO 500 ctDNA
2.6.1 or below
Single-version
No
DRAGEN pipelines**
4.3.6+
Multi-version
Yes
DRAGEN pipelines**
4.2 or below
Single-version
No
The following instructions describe steps to set up a run on NovaSeq 6000Dx Analysis Application.
Use the following steps to configure a TruSight™ Oncology 500 run in Illumina Run Manager:
Go to the "Runs" section of Illumina Run Manager by selecting "Runs" on the left-hand side
Enter sample data manually or by importing a sample sheet
To enter sample data run manually, select “Create Run”
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
On the "Run Settings" screen, enter a run name with the following criteria:
1 - 40 characters.
Alphanumeric characters, underscores, or dashes only.
Unique across all runs on the instrument.
The run name identifies the run from sequencing through analysis.
[Optional] Enter a run description. The run description must have the following criteria:
1 - 50 characters.
Alphanumeric characters or spaces only.
Spaces must be preceded and followed by an alphanumeric character.
Select kit used during library preparation:
TruSight Oncology 500
TruSight Oncology 500 High-Throughput
Index adapter kit will be automatically selected based on the library prep kit selection
[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.
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.
Select lane information. Options include one to four, or all lanes.
Enter a unique sample ID in the sample ID field with the following criteria:
Controls should be added first.
1 - 40 characters.
Alphanumeric characters, underscores, or dashes only.
Underscores and dashes must be preceded and followed by an alphanumeric character.
Select an index set ID for the DNA / RNA library prepared from the sample.
[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.
Use the table on the "Sample Settings" screen to enter additional sample information.
Enter Pair ID with the following criteria:
1 - 40 characters.
Alphanumeric characters, underscores, or dashes only.
Underscores and dashes must be preceded and followed by an alphanumeric character.
Pairs at most one DNA and one RNA samples from the same biological sample from the same individual.
Select Sample Type: DNA or RNA
Enter Sample Feature: Select HRD for DNA samples with HRD probes. For all other samples, leave the field blank.
[Optional] Enter a sample name with the following criteria:
1 - 50 characters.
Alphanumeric characters, dashes, underscores, or spaces.
Spaces, underscores, and dashes must be preceded and followed by an alphanumeric character.
[Optional] Enter a sample description with the following criteria:
1 - 50 characters.
Alphanumeric characters, dashes, underscores, or spaces.
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.
DRAGEN TSO 500 Analysis Software has optional and required fields that are required in addition to general sample sheet requirements. Follow the steps below to create a valid samplesheet.
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.
The analysis fails if the sample sheet requirements are not met.
Use the following steps to create a valid sample sheet.
Download the sample sheet v2 template that matches the instrument & assay run.
In the Sequencing Settings section, enter the following required parameters:
LibraryPrepKits
Required
Accepted values are: TSO500 or TSO500HT
In the BCL Convert Settings section, enter the following required parameters:
SoftwareVersion
Required
The DRAGEN component software version.
TruSight Oncology 500 2.6.0 requires 3.10.17
. To ensure you are using the latest compatible version, refer to the software release notes.
AdapterRead1
Required
If using 8 bp indexes starting with UP or CP (used with TSO 500): AGATCGGAAGAGCACACGTCTGAACTCCAGTCA If using 10 bp indexes with UDP (used with TSO 500 HT): CTGTCTCTTATACACATCTCCGAGCCCACGAGAC Analysis fails if the incorrect adapter sequences are used
AdapterRead2
Required
If using 8 bp indexes starting with UP or CP (used with TSO 500): AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT If using 10 bp indexes with UDP (used with TSO 500 HT): CTGTCTCTTATACACATCTGACGCTGCCGACGA Analysis fails if the incorrect adapter sequences are used
AdapterBehavior
Required
Enter trim
This indicates that the BCL Convert software trims the specified adapter sequences from each read.
MinimumTrimmedReadLength
Required
Enter 35
. Reads with a length trimmed below this point are masked.
MaskShortReads
Required
Enter 35
. Reads with a length trimmed below this point are masked.
In the BCL Convert Data section, enter the following parameters for each sample.
Sample_ID
Required
Must match a Sample_ID listed in the TSO 500 Data section.
Index
Required
Index 1 sequence valid for Index_ID assigned to matching Sample_ID in the TSO 500 Data section.
Index2
Required
Index 2 sequence valid for Index_ID assigned to matching Sample_ID in the TSO 500 Data section.
Lane
Only for NovaSeq 6000 XP, NovaSeq 6000Dx, or NovaSeq X workflows
Indicates which lane corresponds to a given sample. Enter a single numeric value per row. Cannot be empty, i.e the analysis fails if the Lane column is present without a value in each row.
In the TSO 500 Data section, enter the following parameters:
TSO 500 Data Section header changes depending on the deployment:
Standalone DRAGEN Server and ICA with Manual Launch: TSO500S_Data
ICA with Auto-launch: Cloud_TSO500S_Data
Illumina DRAGEN TruSight Oncology 500 (HRD) Analysis Application on NovaSeq 6000Dx: TSO500HRD_Data
Sample_ID
Required
The unique ID to identify a sample. The sample ID is included in the output file names. Sample IDs are not case sensitive. Sample IDs must have the following characteristics:
- Unique for the run.
- 1–40 characters.
- No spaces.
- Alphanumeric characters with underscores and dashes. If you use an underscore or dash, enter an alphanumeric character before and after the underscore or dash. eg, Sample1-T5B1_022515.
- Cannot be called all
, default
, none
, unknown
, undetermined
, stats
, or reports
.
- Must match a Sample_ID listed in the TSO 500 Data section.
- Illumina recommends that the sample ID be based on the pair ID. Example: <Pair_ID>-DNA,<Pair_ID>-RNA.
- Each sample must have a unique combination of Lane (if applicable), sample ID, and index ID or the analysis will fail.
Sample_Type
Required
Enter DNA
or RNA
.
For HRD samples, this parameter must be DNA
.
Pair_ID
Required
A unique ID that links DNA and RNA from the same biological sample from the same individual. Pair ID shares, at most, one DNA and one RNA sample per run. eg, if a Sample_ID is TestSample1-DNA
for DNA and TestSample1-RNA
for RNA, the Pair_ID TestSample1
will link these samples that are on different rows in the sample sheet together.
If the pair ID is associated with more than one DNA or RNA sample, the analysis fails.
Sample_Feature
Required when using HRD add-on kit
Required for HRD enriched samples.
For DNA samples that have undergone HRD enrichment, enter HRD
in this column of the sample sheet. If the sample has not undergone HRD enrichment, leave the field empty.
Sample_Description
Not Required
Sample description must meet the following requirements: - 1–50 characters. - Alphanumeric characters with underscores, dashes and spaces. If you enter a underscore, dash, or space, enter an alphanumeric character before and after. eg, Solid-FFPE_213.
To ensure a successful analysis, follow these guidelines:
Avoid any blank lines at the end of the sample sheet; these can cause the analysis to fail.
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.
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
To auto-launch analysis from the sequencer run folder, ensure the StartsFromFastq and SampleSheetRequested fields are set to FALSE. To auto-launch analysis from FASTQs after BCL Convert auto-launch, StartsFromFastq and SampleSheet Requested fields must be set to TRUE
Refer to [TSO500_Data] Section for this section's requirements.
SoftwareVersion
Not Required
The TSO500S software version
StartsFromFastq
Required
Set the value to TRUE or FALSE. To auto-launch from BCL files, set to FALSE. To auto-launch from FASTQ files after auto-launch of BCL Convert, set to TRUE.
SampleSheetRequested
Required
Set the value to TRUE or FALSE.
To auto-launch from BCL files, set to FALSE. To auto-launch from FASTQ files after auto-launch of BCL Convert, set to TRUE.
Sample_ID
Not Required
The same sample ID used in the Cloud_TSO500S_Data section.
ProjectName
Not Required
The BaseSpace project name.
LibraryName
Not Required
Combination of sample ID and index values in the following format: sampleID_Index_Index2
LibraryPrepKitName
Required
The Library Prep Kit used.
IndexAdapterKitName
Not Required
The Index Adapter Kit used.
GeneratedVersion
Not Required
The cloud GSS version used to create the sample sheet. Optional if manually updating a sample sheet.
CloudWorkflow
Not Required
Ica_workflow_1
Cloud_TSO500_Pipeline
Required
This value is a universal record number (URN . The valid values are: Solid—urn:ilmn:ica:pipeline:8538a5e3-b8d2-469d-baaf-b2164e54cc51#DRAGEN_TruSight_Oncology_500_v2_6_0_2 Solid HRD —urn:ilmn:ica:pipeline:506f136e-980e-427d-ab39-f91654255bea#DRAGEN_TruSight_Oncology_500_HRD_v2_6_0_2
BCLConvert_Pipeline
Required
The value is a URN in the following format: urn:ilmn:ica:pipeline: <pipeline-ID>#<pipeline-name>
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.
Mismatches between the samples and index primers can cause incorrect results due to loss of positive sample identification. Enter sample IDs and assign indexes in the sample sheet before beginning library preparation. Record sample IDs, indexes, and plate well orientation for reference during library preparation.
SoftwareVersion
Required
Enter the IRM iapp software version 2.6.0-2v12ui
Refer to [TSO500S_Data] Section for this section's requirements.
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.
Note that NovaSeq X Series has a different run set up configuration screen than other instrument platforms. TSO 500 does not support multi analysis, and in order to run TSO 500 on NovaSeq X Series, enter the appropriate Read 1, Read 2, Index 1 and Index 2 described in the instructions below.
BaseSpace Run Planning tool cannot generate a valid sample sheet for NovaSeq 6000Dx Analysis Application. Refer to Sample Sheet Requirements page to create a valid sample sheet.
Run Name
Required
Run Name can contain 255 alphanumeric characters, dashes, underscores, periods, and spaces; and must start with an alphanumeric, a dash or an underscore.
Run Description
Optional
Run Description can contain 255 characters except square brackets, asterisks, and commas.
Instrument Platform
Required
Choose from TSO 500 supported instruments:
NextSeq 500/550
NextSeq 1000/2000
NovaSeq 6000/6000Dx
NovaSeq X Series
Secondary Analysis
Required
BaseSpace/Illumina Connected Analytics (to generate sample sheet for cloud analysis)
Local
Read 1
Required on Instrument Platform NovaSeq X Series
Fill with value 101 for TSO 500 analysis
Index 1
Required on Instrument Platform NovaSeq X Series
Fill the value depending on the TSO 500 assay used:
TSO 500 HT: 10
TSO 500: 8
Index 2
Required on Instrument Platform NovaSeq X Series
Fill the value depending on the TSO 500 assay used:
TSO 500 HT: 10
TSO 500: 8
Read 2
Required on Instrument Platform NovaSeq X Series
Fill with value 101 for TSO 500 analysis
Sample Container ID
Optional
Unique Identifier for the container that holds the sample
Note: On NovaSeq X Series, this page is called "Configuration 1". The right hand corner of the UI displays the Read 1, Read 2, Index 1 and Index 2 entered on the previous run settings screen.
Application*
Required
DRAGEN TruSight Oncology 500 Analysis Software - 2.6.0 (with HRD)
DRAGEN TruSight Oncology 500 Analysis Software - 2.6.0
Description
Optional
Optional text field
Library Prep Kit
Required
TruSight Oncology 500
TruSight Oncology 500 High Throughput
Index Adapter Kit
Required
TSO 500:
TruSight Oncology 500 (NovaSeq6000Dx with S1, S2, S4, or SP Flow Cell, NextSeq1000/2000, NovaSeqX Series)
TruSight Oncology 500 (NovaSeq6000, NextSeq)
TSO 500 HT:
TruSight Oncology 500 High Throughput (NovaSeq6000Dx with S1, S2, S4, or SP Flow Cell, NextSeq1000/2000, NovaSeqX Series)
TruSight Oncology 500 High Throughput (NovaSeq6000, NextSeq)
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.
Read Lengths: Read 1 and Read 2
Required Not applicable on NovaSeq X Series
Auto filled with the standard values, but can be optionally overwritten.
Override Cycles
Required on NovaSeq X Series
Entered based on Run Settings read lengths & index 1 / index 2
Lane Usage
Not applicable on NovaSeq X Series or NextSeq 1000 / 2000
Checkbox allows users to apply the same lane across samples.
Lane
Required if Lane Usage is unchecked Not applicable on NextSeq 1000 / 2000
Specify lanes for each sample. The unmarked checkbox at the top of the dropdown selects all lanes.
Pair ID
Required
The identifier used to pair DNA and RNA samples in a run. The field is mandatory whether a sample is part of a pair, or not.
To note: The Sample ID field in the generated samplesheet will be auto-filled based on the Pair ID values captured. “_dna” and “_rna” (for DNA and RNA samples respectively) will be appended to the Pair ID value to create the Sample ID.
DNA Index ID
Required
Index set ID options are based on selected Index Adapter Kit
DNA Sample Feature
Required for TSO 500 HRD
Column appears when TSO 500 HRD application is selected. Enter for HRD enriched DNA Samples
RNA Index ID
Required
Index set ID options are based on selected Index Adapter Kit
Project
Optional
Optional field to describe the associated project
Starts from Fastq
Required
True or False
If auto-launching TSO 500 from BCL files, set the value to False. If auto-launching TSO 500 from FASTQ after auto-launching BCL Convert, set the value to True.
DNA Barcode Mismatches Index 1**
DNA Barcode Mismatches Index 2**
RNA Barcode Mismatches Index 1**
RNA Barcode Mismatches Index 2**
Required on NovaSeq X
Default value is set to 1.
These fields are required by NovaSeq X and represent BCL Convert settings for index diversity checks when demultiplexing. These values are not used in TSO 500 analysis.
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.
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.
Please review these guided examples of analysis workflows that include a step of setting up a run in BaseSpace Run Planning tool:
Sample Sheet templates for TSO 500 v2.6.0 standalone DRAGEN server and ICA manual launch analysis can be found in the table below. For auto-launch compatible sample sheets, use BaseSpace Run Planner.
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.
For interactive run planning or to create a sample sheet for ICA Autolaunch, use BaseSpace Run Planner to create valid sample sheets for either local or cloud analysis. To set up a run in BaseSpace run planner, refer to Sample Sheet Creation in BaseSpace Run Planner.
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:
TSO500
NextSeq 550
Standard
TSO500 + HRD
NextSeq 550
Standard
TSO500 + HRD
NovaSeq 6000
Standard
TSO500 + HRD
NovaSeq 6000Dx (in RUO mode)
Standard
TSO500 HT
NextSeq 1000 / 2000
Standard
TSO500 HT
NovaSeq 6000
Standard
TSO500 HT
NovaSeq 6000
XP*
TSO500 HT
NovaSeq 6000Dx (in RUO mode)
Standard
TSO500 HT
NovaSeq 6000Dx (in RUO mode)
XP*
TSO500 HT
NovaSeq X
Standard
TSO500 HT
NovaSeq X
XP*
*Lane numbers cannot exceed what is supported by the flow cell in use.
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.
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).
TruSight Oncology 500 HRD is not available in Japan
Single nucleotide variants (SNVs)
Insertions
Deletions
Copy number variants (CNVs)
Exon-level CNVs
Multinucleotide variants (MNVs)
Genomic Instability Score (GIS Score) *
Tumor mutational burden (TMB)
Microsatellite instability (MSI)
Fusions
Splice variants
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.
*Requires TruSight Oncology 500 HRD add-on kit
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.
DRAGEN TruSight Oncology 500 analysis software is compatible with data generated on the Illumina instruments as summarized in the table below.
NextSeq 550Dx (RUO mode)
Yes
Yes
No
N/A
NextSeq 500/550
Yes
Yes
N/A
N/A
NovaSeq 6000
Yes
Yes
N/A
N/A
NovaSeq 6000Dx (RUO mode)
Yes
Yes
Yes
N/A
NextSeq 1000/2000
Yes
Yes
N/A
No
NovaSeq X
Yes
Yes
N/A
No
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.
Before running the analysis, confirm that the output directory for the software to write to is empty and does not include results of previous analyses.
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:
NextSeq 500/550/550Dx (RUO) HO flow cell
32-55
82-85
150
NovaSeq 6000/6000Dx (RUO) SP Flow Cell
85-100
250-374
300
NovaSeq 6000/6000Dx (RUO) S1 Flow Cell
164-200
360-665
800
NovaSeq 6000/6000Dx (RUO) S2 Flow Cell
290-460
890-1600
1500
NovaSeq 6000/6000Dx (RUO) S4 Flow Cell
800-1200
2700-4100
3000
NovaSeq X 1.5B
213
352
800
NovaSeq X 10B
1100
1800
3000
NovaSeq X 25B
1800
3300
4000
NextSeq 1000/2000
41
107
150
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.
Moving or modifying files during an analysis may cause the analysis to fail or provide incorrect results.
When the analysis run completes, the DRAGEN TruSight Oncology 500 Analysis Software generates an analysis output folder in a specified location.
To view analysis output, navigate to the analysis output folder and select the files that you want to view.
Single output folder structure is as follows.
Logs_Intermediates
AdditionalSarjMetrics— Contains per pair ID calculations to support the PCT_TARGET_250X metric.
Annotation—Contains outputs for small variant annotation.
Subfolders per sample ID—Contains the aligned small variants JSON.
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 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.
This section describes each output folder generated during analysis and where to find metric and analytic files when the pipeline is executed. The same output folder structure and content exist in ICA and BaseSpace Sequence Hub.
Run ID
TSO500_Nextflow_logs
_manifest.json
Results
_tags.json
Logs_intermediates
Errors—This folder is only present when analysis fails
The TSO_500_Nextflow_Logs provides information related to the execution of the pipeline on ICA as a whole and for specific nodes (when an analysis is split across multiple nodes). It contains files used to execute parts of the workflow on different nodes as well as records of the nextflow execution on those nodes.
TSO_500_Nextflow_Logs
_manifest.json
Contains the aggregated MetricsOutput.tsv file at the root level. Additionally, the Results folder contains a subfolder for each pair ID.
Results
MetricsOutput.tsv
Sample_1
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
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
Contains Errors.tsv. This file contains the summary of all the errors encountered during pipeline execution.
Errors
Errors.tsv
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:
On the “Completed” runs tab, select the run
Review the run details page, and this will give the information to access the output folder
External Location: is the input for the run
Analysis Output Folder: is where the output is stored. To navigate to this page, follow the “server location” and the gds analysis output folder
Navigate to the directory that contains the analysis output folder
Open the folder, and then select the files that you want to view
The installation script for DRAGEN TruSight Oncology 500 Analysis Software installs the following software and dependencies:
DRAGEN TruSight Oncology 500 Analysis Software itself
DRAGEN Software if a compatible version is not present
Docker software if a compatible version is not present
A script required to generate DRAGEN genome hash table
A script to check that DRAGEN TruSight Oncology 500 Analysis Software is installed properly
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).
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
DRAGEN TruSight Oncology 500 v2.6.0 Analysis Software is not compatible with DRAGEN Software v4.0 or above on the same standalone DRAGEN server.
TSOCombined
license
TSO500_HRD
license (to analyze data generated with the TSO 500 HRD add-on kit)
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.
DRAGEN TruSight Oncology 500 Analysis Software v2.6.0 can be installed on one DRAGEN server with:
DRAGEN TruSight Oncology 500 ctDNA Analysis Software v2.6.0 (v3.10.17*)
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*))
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.
When installing DRAGEN TruSight Oncology 500 and DRAGEN TruSight Oncology 500 ctDNA software on the same DRAGEN server, install the software with the highest corresponding DRAGEN Software version last, as versions below v2.6.0 will overwrite with its corresponding DRAGEN Software version.
If a prior version of DRAGEN TruSight Oncology 500 Analysis Software (eg. v2.5.3) is installed after v2.6.0, re-execute the installation script for v2.6.0 to install the compatible version of DRAGEN Software without impacting other installations.
As a root user, perform the following steps to install DRAGEN TruSight Oncology 500 v2.6.0 Analysis Software:
Download the installation package provided in the email from Illumina. The link expires after 7 days.
It is recommended to use a command line tool like wget or curl to download the file rather than pasting the link into the web browser bar. For example:
curl -o {filename} "{link}"
wget -O {filename} '{link}'
Where the file name is the installation script file name, and the link is provided by Illumina Customer Care.
Make sure no other analysis is being performed. Installing the software while performing other analyses prevent the installer process from proceeding
Copy the install script to the /staging
directory to store the script in the directory.
Installation Script: install_DRAGEN_TSO500-2.6.0.run
MD5sum: 578cda2b8837845b26e2c3c020f2264c
Use the following command to update the run script permission:
chmod +x /staging/install_DRAGEN_TSO500-2.6.0.run
Use the following command to run the installation script, which runs for approximately 20 minutes:
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.
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.
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.
Log out of the server and then log back in.
To install a license (TSOCombined
and/or TSO500_HRD
) on a DRAGEN server connected to the internet:
Confirm that the server is connected to the Internet, example: ping www.illumina.com
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:
Download and save the license file(s) to a location that is accessible from the DRAGEN server
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.
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.
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:
Determine how many DRAGEN servers are available to run.
Run demultiplexing on a single DRAGEN server.
Moving or modifying files during an analysis may cause the analysis to fail or provide incorrect results.
To sequence runs on multiple DRAGEN servers using the NovaSeq 6000 XP workflow, modify the sample sheet to include a subset of the lanes. For example, on an S2 flowcell, create two modified sample sheets with one containing the samples from lane 1 and the other from lane 2. This allows only the sample sheet to be modified instead of copying files between servers. This strategy would use the start from Run Folder commands without the --demultiplexOnly
option. The entire run folder would need to be copied to each analysis server as demultiplexing is performed once per server.
Transfer the FASTQ folder output from the original DRAGEN server to additional servers.
Logs_Intermediates/FastqGeneration.
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.
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.
*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.
Use BaseSpace Sequence Hub Run Planning tool or the sample sheet templates provided on the support page to create and export a sample sheet.
If BaseSpace Run Planning tool is not available in your region, use the sample sheet template.
Data is uploaded to BaseSpace Sequence Hub and then pushed to ICA. You can monitor the run in BaseSpace Sequence Hub.
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
If necessary, you can requeue the analysis via BaseSpace Sequence Hub.
View the analysis output results in either BaseSpace Sequence Hub or ICA.
To avoid invalid sample sheet configurations, Illumina recommends using BaseSpace Run Planning tool to generate sample sheets. Using an invalid sample sheet can result in failed runs and analyses.
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.
You can requeue analysis of a run via the run's Summary page in BaseSpace Sequence Hub.
Please review these guided examples of using DRAGEN TSO 500 Analysis Software with auto-launch on ICA:
File name: {SAMPLE_ID}_hard-filtered.gvcf.gz
The small variant genome variant call file contains information on all candidate small variants evaluated, including complex variants up to 15 bp from phased variant calling across the entire TSO 500 panel.
The variant status is determined by the FILTER column in the genome VCF as follows.
File name: {SAMPLE_ID}_DNAVariants_Annotated.json.gz
The small variants annotated file provides variant annotation information for all nonreference positions from the genome VCF including pass and nonpass variants.
The TMB trace file provides comprehensive information on how the TMB value is calculated for a given sample. All passing small variants from the small variant filtering step are included in this file. To calculate the numerator of the TmbPerMb value in the TMB JSON, set the TSV file filter to use the IncludedInTMBNumerator with a value of True.
The TMB trace file is not intended to be used for variant inspections. The filtering statuses are exclusively set for TMB calculation purposes. Setting a filter does not translate into the classification of a variant as somatic or germline.
The copy number VCF file contains CNV calls for DNA libraries of the amplification genes targeted by DRAGEN TruSight Oncology 500 Analysis Software. The CNV call indicates fold change results for each gene classified as reference, deletion, or amplification.
The value in the QUAL column of the VCF is a Phred transformation of the p-value where Q=-10xlog10(p-value). The p-value is derived from the t-test between the fold change of the gene against the rest of the genome. Higher Q-scores indicate higher confidence in the CNV call.
In the VCF notation, <DUP> indicates the detected fold change (FC) is greater than a predefined amplification cutoff. <DEL> indicates the detected FC is less than a predefined deletion cutoff for that gene. This cutoff can vary from gene to gene.
In analysis versions prior to v2.5, <DEL> calls in the VCF are marked as LowValidation. The LowValidation filter indicates that the calls have been validated only with in silico data sets and are provided as information only.
Each copy number variant is reported as a fold change on normalized read depth in a testing sample relative to the normalized read depth in diploid genomes. Given tumor purity, you can infer the ploidy of a gene in the sample from the reported fold change.
Given tumor purity X%, for a reported fold change Y, you can calculate the copy number n using the following equation:
For example, a tumor purity at 30% and a MET with fold change of 2.2x indicates that 10 copies of MET DNA are observed.
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
.
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
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"
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:
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.
Use BCL Convert to produce FASTQ files for DRAGEN TruSight Oncology 500 Analysis Software. Using bcl2fastq does not produce the same results and is discouraged.
Make sure that BCL Convert is set to write UMI sequences to the read headers in the FASTQ files.
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.
TSOCombined
license has been pre-installed to DRAGEN servers in manufacturing since August 2022 and TSO500_HRD
since February 2025 and additionally distributed to DRAGEN servers connected online. To generate a list of installed DRAGEN server licenses, run the following command: /opt/edico/bin/dragen_lic
. If a license is not installed, contact Illumina Customer Care at for the license.
A non-root user must be a member of the Docker group to run Docker. For more information on Docker permission requirements and alternatives to running as root, refer to the Docker documentation available on the .
Contact Illumina Customer Care at to obtain the DRAGEN TruSight Oncology 500 Analysis Software installer package.
Use the following command to build the DRAGEN server hash table, which runs for approximately 60 minutes:
/usr/local/bin/build-hashtable_DRAGEN_TSO500-2.6.0.sh
Refer to if any errors occur.
Review license requirements, how to check which licenses are installed and how to receive a license in . Licenses can be installed before or after DRAGEN TSO 500 software installation.
Contact Customer Care at to request a license file for each of the needed licenses
Import the sample sheet to the instrument and start the sequencing run. Refer to for sample sheet guidance.
Refer to the for information on setting up a BaseSpace Sequence Hub project.
Refer to the for more information on requeuing an analysis.
Refer to the for information on how to manage accounts and subscriptions.
Refer to for more information.
The FASTQ folder structure conforms to the folder structure in
—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.
Docker
20.10 or greater
Docker 20.10.15
DRAGEN Software
v3.10.x where x is 17 or greater
DRAGEN Software 3.10.17
Demultiplexing
DRAGEN_TSO500_2.6.0.sh --resourcesFolder /staging/illumina/DRAGEN_TSO500/resources --hashtableFolder /staging/illumina/DRAGEN_TSO500/ref_hashtable --runFolder /staging/{RunFolderName} --analysisFolder /staging/{DemultiplexAnalysisFolderName} --demultiplexOnly --sampleSheet /staging/illumina/{SampleSheetName}
Analysis (one server)
DRAGEN_TSO500_2.6.0.sh --resourcesFolder /staging/illumina/DRAGEN_TSO500/resources --hashtableFolder /staging/illumina/DRAGEN_TSO500/ref_hashtable --fastqFolder /staging/{DemultiplexAnalysisFolderName}/Logs_Intermediates/FastqGeneration/ --analysisFolder /staging/{Node1AnalysisFolderName} --sampleSheet /staging/illumina/{SampleSheetName} --sampleOrPairIDs Pair_1,Pair_2
Analysis (additional servers)
DRAGEN_TSO500_2.6.0.sh --resourcesFolder /staging/illumina/DRAGEN_TSO500/resources --hashtableFolder /staging/illumina/DRAGEN_TSO500/ref_hashtable --fastqFolder /staging/{DemultiplexAnalysisFolderName}/Logs_Intermediates/FastqGeneration/ --analysisFolder /staging/{Node1AnalysisFolderName} --sampleSheet /staging/illumina/{SampleSheetName} --sampleOrPairIDs Pair_3
Gather
DRAGEN_TSO500_2.6.0.sh --analysisFolder /Gathered_Results --resourcesFolder staging/illumina/DRAGEN_TSO500/resources --runFolder /staging/{RunFolderName}/--sampleSheet /staging/illumina/{SampleSheetName} --gather /Demultiplex_Output /Node1_Output /Node2_Output
NextSeq 500/550/550Dx (RUO) HO flow cell
350
NovaSeq 6000/6000Dx (RUO) SP Flow Cell
500
NovaSeq 6000/6000Dx (RUO) S1 Flow Cell
1100
NovaSeq 6000/6000Dx (RUO) S2 Flow Cell
2500
NovaSeq 6000/6000Dx (RUO) S4 Flow Cell
4300
NovaSeq X 1.5B
2000
NovaSeq X 10B
4300
NovaSeq X 25B
8400
NextSeq 1000/2000
350
PASS
PASS variants.
base_quality
Site filtered because median base quality of alt reads at this locus does not meet threshold.
filtered_reads
Site filtered because the fraction of reads is too large.
fragment_length
Site filtered because absolute difference between the median fragment length of alt reads and median fragment length of ref reads at this locus exceeds threshold.
low_depth
Site filtered because the read depth is too low.
low_frac_info_reads
Site filtered because the fraction of informative reads is below threshold.
long_indel
Site filtered because the indel length is too long.
mapping_quality
Site filtered because median mapping quality of alt reads at this locus does not meet threshold.
multiallelic
Site filtered because more than two alt alleles pass tumor LOD.
no_reliable_supporting_read
Site filtered because no reliable supporting somatic read exists.
read_position
Site filtered because median of distances between start/end of read and this locus is below threshold.
str_contraction
Site filtered due to suspected PCR error where the alt allele is one repeat unit less than the reference.
too_few_supporting_reads
Site filtered because there are too few supporting reads in the tumor sample.
weak_evidence
Somatic variant score (SQ) does not meet threshold.
systematic_noise
Site filtered based on evidence of systematic noise in normal sample.
excluded_regions
Site overlaps with VC excluded regions bed.
Chromosome
Chromosome
Position
Position of variant
RefCall
Reference base
AltCall
Alternate base
VAF
Variant allele frequency
Depth
Coverage of position
CytoBand
Cytoband of variant
GeneName
Name of gene if applicable. A semicolon delimited list is used for multiple genes.
VariantType
Type of the variant: SNV, insertion, deletion, MNV
CosmicIDs
Cosmic IDs, if multiple concatenated by “;”
MaxCosmicCount
Maximum Cosmic study count
AlleleCountsGnomadExome
Variant allele count in gnomAD exome database
AlleleCountsGnomadGenome
Variant allele count in gnomAD genome database
AlleleCounts1000Genomes
Variant allele count in 1000 genomes database
MaxDatabaseAlleleCounts
Maximum variant allele count over the three databases
GermlineFilterDatabase
TRUE if variant was filtered by the database filter
GermlineFilterProxi
TRUE if variant was filtered by the proxi filter
CodingVariant
TRUE if variant is in the coding region
Nonsynonymous
TRUE if variant has any transcript annotations with nonsynonymous consequences
IncludedinTMBNumberator
TRUE if variant is used in the TMB calculation
--help
No
Displays a help screen with available command line options.
--analysisFolder
No
Path to the local analysis folder. The default location is /staging/DRAGEN_TSO500_2.6.0_Analysis_{timestamp}
. If not using the default location, provide the full path to the local analysis folder. Folder must have sufficient space and must be on an NVMe SSD drive. For example, the /staging
directory on the DRAGEN server.
Refer to table in Storage Requirements for minimum disk space requirements.
--resourcesFolder
No
Path to the resource folder location. The default location is /staging/illumina/DRAGEN_TSO500_2.6.0/resources
. If not using the default location, enter the full path to the resource folder.
--runFolder
Yes
Required when --fastqFolder
is not specified. Provide the full path to the local run folder.
--fastqFolder
Yes
Required when --runFolder
is not specified. Provide the full path to the local FASTQ folder. Analysis starts at this location.
--user
No
Optional for Docker. Specify the user ID to be used within the Docker container.
--version
No
Displays the version of the software.
--sampleSheet
No
Provide the full path, including file name, if not provided as SampleSheet.csv
in the run folder
--sampleOrPairIDs
No
Provide the comma-delimited sample or pair IDs that should be processed on this node with no spaces. For example, Pair_1,Pair_2,Sample_1.
--demultiplexOnly
No
Demultiplex to generate FASTQ only without additional analysis.
--gather
No
Follow this option for any directories with results that should be gathered into a single Results folder.
--hashtableFolder
No
Defaults to the DRAGEN hash table location created upon install. If not using the default location, enter the hash table location.
Config folder
Configuration files
Data folder
*.bcl files
Images folder
[Optional] Raw sequencing image files.
Interop folder
Interop metric files.
Logs folder
[Optional] Sequencing system log files.
RTALogs folder
Real-Time Analysis (RTA) log files.
RunInfo.xml file
Run information.
RunParameters.xml file
Run parameters.
SampleSheet.csv file
Sample information. If you want to use a sample sheet that is not in the run folder or a sample sheet named something other than SampleSheet.csv
, provide the full path.
Refer to RNA Analysis Methods for more information.
The splice variant VCF contains all candidate splice variants targeted by the analysis panel identified by the RNA analysis pipeline. You can apply the following filters for each variant call:
LowQ
Splice variant score < passing quality score threshold value of 1.
PASS
Splice variant score ≥ passing quality score threshold value of 1.
LowUniqueAlignments
All splice junction supporting reads map to a unique genomic interval near at least one of the two splice sites.
Refer to the headers in the output for more information about each column.
If available, each splice variant is annotated using the Illumina Annotation Engine. The following information is captured in the JSON:
HGNC Gene
Transcript
Exons
Introns
Canonical
Consequence
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
DOUBLE_BROKEN_EXON
Confidence filter
If both breakpoints are distant from annotated exon boundaries, the number of supporting reads do not satisfy a high threshold requirement (≥ 10 supporting reads).
LOW_MAPQ
Confidence filter
All fusion supporting read alignments at either of the breakpoints have MAPQ < 20.
LOW_UNIQUE_ALIGNMENTS
Confidence filter
All fusion supporting read alignments map to a unique genomic interval at either of the breakpoints.
LOW_SCORE
Confidence filter
The fusion candidate has probabilistic score as determined by the features of the candidate.
MIN_SUPPORT