Command-Line Options

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.

Option	Required	Description
--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_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/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.

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.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.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.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.sh \ --fastqFolder /staging/{FastqFolderName} \ --analysisFolder /staging/{AnalysisFolderName} \ --sampleOrPairIDs "Pair_1,Pair2"

Starting from BCL Files

For the data generated by NextSeq 1000/2000 and NextSeq X, the analysis can only be started from FASTQs and not from BCLs.

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:

Folder/File	Description
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.

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 FASTQ folder structure conforms to the folder structure in FASTQ File Organization.

• 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.

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.