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

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

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

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

When the auto-launched analysis completed, you will see the analysis result shows results from both the BCL Convert pipeline and the TSO 500 ctDNA pipeline, like below:

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 ctDNA 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_CTDNA-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_CTDNA.sh \ --runFolder /staging/{RunFolderName} \ --analysisFolder /staging/{AnalysisFolderName} \

Starting from BCL Files

If starting from BCL (*.bcl) files, DRAGEN TruSight Oncology 500 ctDNA 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 ctDNA Analysis Software using FASTQ (*.fastq) files. The requirements apply to Docker.

• Full path to an existing FASTQ folder.

• The FASTQ folder structure conforms to the folder structure in

• 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 ctDNA 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.

Start the DRAGEN TruSight Oncology 500 ctDNA Analysis Software with the DRAGEN_TSO500_CTDNA-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 . 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

Methods for Launching Analysis

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

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

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

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

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 ctDNA analysis auto-launch. For information on preparing your instrument for DRAGEN TSO 500 ctDNA Auto-launch, refer to the documentation for your instrument.

Input Validation Steps

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

• Resource bundle integrity

• DRAGEN license validity

• NEW in v2.6.3! Instrument Type determination

Instrument type is required by the software because different instrument platforms produce output with distinct systematic noise profiles. DRAGEN TSO 500 ctDNA analysis software requires instrument-specific baseline noise files. Before kicking off analysis, the software determines the instrument type in order to select which baseline files to use. This means that even when starting from FASTQ, all samples in the run must be sequenced on the same type of instrument.

Instrument Type Determination for Baselines

When starting analysis from BCL files, the software references the RunInfo.xml file in the Run folder to identify the sequencing instrument type. For analyses beginning with FASTQ files, the software reads the FASTQ file headers for this information.

The Combined Variant Output File will include the instrument type

Systematic Noise files for certain variant types depend on library prep kit, instrument type, or both. The software will select the correct set of resource files (systematic noise, panel of normals) according to its determination of instrument type and library prep kit. VCF headers contain details on which resource files were used in the analysis.

• Library Preparation Kit: TSO500 ctDNA v2 (index length: 10)

  • Instrument Types:

    • NovaSeq 6000, NovaSeq 6000Dx: Use one set of baselines.

How to Launch Analysis

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

1. Edit Project and Add Bundle: Edit the Project and add the bundle titled, "DRAGEN TSO 500 ctDNA v2.6.0 (XX)." XX is a 2-letter code designating the region from which you are launching the analysis. Adding the Bundle automatically adds the pipeline and associated resource files and datasets to the Project. For information on Bundles, refer to the Bundles section in .

1.  Upload the sequencing data: For information on viewing and uploading data, refer to the Data section in .

2. Start Analysis: In the Project, navigate to Pipelines, select the TSO 500 ctDNA v2.6.0 Pipeline, and then select  "Start New Analysis". Set up the new analysis by configuring the parameters listed in the . When the required files are completed, start analysis.

3. Download Results: After analysis is complete, navigate to results in the configured output location.

Please see the Illumina Support Shorts for guidance on how to set up and run DRAGEN TSO 500 RUO analysis on ICA. This is the same process as DRAGEN TSO 500 ctDNA, but with different inputs specific to DRAGEN TSO 500 solid:

Analysis Parameters on ICA

To launch an analysis via the ICA user interface, configure a DRAGEN TSO 500 ctDNA pipeline analysis with the following parameters.

Known Limitations

• FASTQ Folder Naming Requirements

  • When specifying input FASTQ folder names, avoid using folder names that consist entirely of numeric characters with a leading zero, as this will cause the software to error out.

  • Unsupported naming pattern:

For information about using pipelines, refer to .