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.

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 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.

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.