The DRAGEN microsatellite instability status (MSI) module assesses microsatellite sites for evidence of microsatellite instability, relative to a set of baseline normal samples that are based on Jensen– Shannon (JS) distance (an information entropy metric). In total, there are 2343 selected MSI sites with 6 or 7 single nucleotide repeats in the panel. For MSI sites with 500 or more spanning duplex collapsed reads, JS distance is calculated using a test sample vs baseline normal samples, and then any two baseline normal samples. If the JS distance is significantly higher in the test sample vs baseline normal with p-value ≤ 0.01 and the JS distance difference is ≥ 0.02, the site is considered unstable. The final MSI score aggregates all JS distance across all unstable sites. The input is the BAM file from the DNA alignment and read collapsing step, and the output is an MSI metric file.

Sequencing data stored in BCL format are demultiplexed through a process that uses the index sequences unique to each sample to assign clusters to the library from which they originated. Each cluster contains two indexes (i7 and i5 sequences, one at each end of the library fragment). The combination of those index sequences are used to demultiplex the pooled libraries.

After demultiplexing, this process generates FASTQ files, which contain the sequencing reads for each individual sample library and the associated quality scores for each base call, excluding reads from any clusters that did not pass filter.

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.

The Run Metrics section of the metrics output report provides sequencing run quality metrics along with suggested values to determine if they are within an acceptable range. The overall percentage of reads passing filter is compared to a minimum threshold. For Read 1 and Read 2, the average percentage of bases ≥ Q30, which gives a prediction of the probability of an incorrect base call (Q‑score), are also compared to a minimum threshold. The following tables show run metric and quality threshold information for different systems.

The values in the Run Metrics section are listed as NA in the following situations:

• If the analysis was started from FASTQ files.

• If the analysis was started from BCL files and the InterOp files are missing or corrupt.

NovaSeq 6000 or NovaSeq 6000Dx (RUO)

NovaSeq X

Use the following steps to configure a TruSight™ Oncology 500 ctDNA run in Illumina Run Manager.

1. Go to the "Runs" section of Illumina Run Manager by selecting "Runs" on the left-hand side.

2. Enter sample data manually or by importing a sample sheet

File name: {SampleID}_CombinedVariantOutput.tsv

The combined variant file contains the variants and biomarkers in a single file. The output contains the following variant types and biomarkers:

• Small variants (including EGFR complex variants)

• Copy number variants

The maximum somatic variant allele frequency (MSAF) is the highest VAF of a confirmed somatic mutation. The MSAF is often used as a surrogate for tumor fraction, especially when the ctDNA tumor fraction is high.

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

DNA Alignment and Read Collapsing

The alignment step uses DRAGEN Aligner with UMI collapsing to align DNA sequences in FASTQ files to the hg19_decoy genome. This step combines sets of reads (ie, families) that are grouped together based on genomic locations and UMI tags into representative sequences. This process accurately removes duplicate reads and sequencing errors without losing the signal of very low frequency (< 1%) sequence variations.

This alignment step generates BAM files (*.bam) and BAM index files (*.bam.bai) that are saved to the alignment folder. A BAM file is the compressed binary version of a SAM file that is used to represent aligned sequences.

Read collapsing adds the following BAM tags:

• RX/XU: UMI combination. RX is duplicated from XU to satisfy the BAM/SAM format

• XV: Number of reads in the family on one strand.

• XW: Number of reads in the duplex-family or 0 if not a duplex family.

Indel Realignment and Read Stitching

The Gemini software component performs local indel realignment, paired‑read stitching, and read filtering. A stitched read is a single read that has been combined from a pair of reads. Reads near detected indels are realigned to remove alignment artifacts. The input is a single BAM file and the reference genome FASTA used to align it; the output is a corresponding single BAM file with stitched, pair‑realigned reads. Read pairs with poor map quality or supplementary and secondary alignments from the input BAM are ignored.

The following BAM tags are added to the stitched reads:

• XD—Directional support string indicating forward, reverse, and stitched positions.

• XR—Read pair orientation, which can be either forward-reverse (FR) or reverse-forward (RF).

The gene and exon coverage report files are tab-separated value (TSV) files with coverage values matching respectively the exons and genes specified in the manifest file.

The Illumina DRAGEN TruSight Oncology 500 (TSO 500) ctDNA Analysis Software supports analysis for DNA libraries that are isolated from plasma and prepared using TruSight Oncology 500 ctDNA v2 and v1 assays. The software, which can be run on the DRAGEN Server, on Illumina Connected Analytics, and as an analysis application on NovaSeq 6000Dx, produces a variant call file (VCF) for small variants. Other outputs include tumor mutational burden (TMB), a Jensen-Shannon divergence (sum JSD) score that can be used for evaluating microsatellite instability (MSI) status, files with copy number variants (CNV), fusions as well as coverage reports.

The secondary analysis software starts from a sequencing run folder containing base call files (BCL) or from FASTQ files staged in a FASTQ folder.

This document provides information on installation, configuration, running, troubleshooting as well as analysis algorithms of DRAGEN TruSight Oncology 500 ctDNA analysis software on Illumina Connected Analytics, standalone DRAGEN server, and the NovaSeq 6000Dx analysis application.

The software is optimized for analyzing sequencing outputs generated by the TruSight Oncology 500 ctDNA v2 and v1 assays. Modification might lead to inaccurate data and is a violation of the Illumina Software Subscription Agreement.

Scope

This resource provides information on installation, configuration, running, troubleshooting and analysis algorithms for the following software:

The content is applicable to all 3 software versions unless otherwise specified. The content related to setting up and running the analysis on ICA is only relevant to software versions v2.6.0 and v2.6.1. The content related to the analysis application on NovaSeq 6000Dx is only relevant to v2.6.1.

Local and Cloud Deployments

Local analysis is available using a standalone DRAGEN server or an application with a user interface on NovaSeq 6000Dx. The software on the standalone DRAGEN server allows for analysis on a single DRAGEN server or splitting across multiple servers.

Cloud analysis is available on Illumina Connected Analytics with auto-launch or manual launch. Both methods are available from BCLs and FASTQs.

Instrument Compatibility

DRAGEN TruSight Oncology 500 ctDNA analysis software is compatible with data generated on the Illumina instruments as summarized in the table below.

Navigation of Guide

This resource provides information on installation, configuration, running, troubleshooting as well as analysis algorithms of DRAGEN TruSight Oncology 500 ctDNA analysis software on Illumina Connected Analytics, standalone DRAGEN server, and the NovaSeq 6000Dx analysis application.

Workflow Diagram

File Name: MetricsOutput.tsv

The metrics output file is a final combined metrics report that provides sample status, key analysis metrics, and metadata in a tab-separated values (TSV) file. Sample metrics within the report indicate guideline‑suggested lower specification limits (LSL) and upper specification limits (USL) for each sample in the run.

One metrics output file is generated for the entire run. An additional file is generated for each sample.

Run Metrics

Run metrics from the analysis module indicate the quality of the sequencing run.

Review the following metrics to assess run data quality:

The values in the Run Metrics section are listed as NA in the following situations:

• The analysis was started from FASTQ files.

• The analysis was started from BCL files and the InterOp files are missing or corrupt.

• [NovaSeqX Plus only] There is no PCT_PF_READS value in NovaSeqX Plus runs, so the PCT_PF_READS value will always be NA.

Sample QC Metrics

Review the following metrics to assess sample data quality:

*The recommended threshold of 0.059 for GENE_SCALED_MAD only applies to real cell‑free DNA.

For troubleshooting information, refer to

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.

The contamination score evaluates presence of sample-to-sample contamination. The algorithm uses common germline SNPs in the homozygous state expected to have variant allele frequencies (VAF) at 0% and 100%. In contaminated samples, the VAFs shift away from the expected values allowing the detection of sample-to-sample contamination.

Contamination Score Calculation

The contamination score is calculated using the SNP error file and Pileup file that are generated during the small variant calling, as well as the TMB trace file. The algorithm includes the following steps:

• All positions that overlap with a pre-defined set of common SNPs that have variant allele frequencies of < 25% or > 75% are collected (only SNP are considered, indels are excluded)

• Variants in CNV events are removed using a clustering method

• The likelihood that the positions are an error or a real mutation is calculated by:

CONTAMINATION_SCORE = sum(log10(P(v_i is False Positive)))

Contamination Score Interpretation

• The contamination score is output in the metrics output file, MetricsOutput.tsv

• If a contamination score is equal or below 1227 (the upper specification limit provided in the "USL Guideline" field in the metrics output file, see ), the sample has less than 0.4% sample-to-sample contamination.

• If a contamination score is above 1227, the sample has more than 0.4% sample-to-sample contamination. In this case, an estimation of the contamination can be obtained from the PCT_CONTAMINATION_EST metric, see more details on the . As noted, PCT_CONTAMINATION_EST is not valid unless the contamination score exceeds 1227.

• Visual examination can help determine if a shift of VAFs is due to true contamination

How to build a VAF plot for visual examination

1. To build a VAF plot, use the {Sample_ID}.tmb.trace.csv file. Filter to only germline variants (for example, by using tags "Germline_DB" and "Germline_Proxi" in the column "Status") and use values in the VAF column.

2. Select Scatter from the Charts menu

3. Review plot as described above analyzing whether variants are scattered or clustered around 50% and 100% VAF

Prerequisites

Illumina Connected Analytics (ICA) subscription includes access to DRAGEN TruSight Oncology 500 ctDNA Analysis Software. To get started, you need:

• An ICA account with a valid subscription

• A positive balance of iCredits for data storage

Refer to the for information on how to register ICA subscription and iCredits.

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

Overview

A sample sheet is required for each analysis with DRAGEN TruSight Oncology 500 ctDNA 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 ctDNA Analysis Software is compatible with the sample sheet v2. For general information on the sample sheet v2, refer to .

The sample sheet includes a list of samples and their index sequences, along with additional information required to run DRAGEN TruSight Oncology 500 ctDNA Analysis Software. For example, the library prep kit used for analysis will need to be listed in the sample sheet. Appropriate index adapter sequences are determined by the assay used to perform analysis.

DRAGEN TruSight Oncology 500 uses QC metrics to assess the validity of analysis for DNA libraries that pass contamination quality control. If the library fails one or more quality metrics, then the corresponding variant type or biomarker is not reported, and the associated QC category in the report header displays FAIL.

DNA library QC results are available in the MetricsOutput.tsv file. Refer to for details.

The software processes sequencing data to perform quality control, detect variants, determine tumor mutational burden (TMB), microsatellite instability (MSI) status, and report results. The following sections describe the analysis methods used in DRAGEN TruSight Oncology 500 ctDNA Analysis Software.

DRAGEN TruSight Oncology 500 ctDNA Analysis Software uses the following workflows to analyze sequencing data.

• FASTQ Generation

• DNA Analysis

  • Contamination Detection

  • Copy Number Variant (CNV) Calling

  • DNA Alignment and Read Collapsing

  • Fusion Calling

  • Fusion Filtering

  • Indel realignment and read stitching

  • Max Somatic AF

  • Microsatellite Instability (MSI) Status

  • Phased Variant Calling

  • Small Variant Calling

  • Small Variant Filtering

  • Tumor Mutational Burden (TMB) Scoring

  • Variant Merging

• Quality Control

  • Run QC

  • DNA Sample QC

Introduction

The NovaSeq X platform supports loading samples from different assays into different lanes within a single sequencing run. To ensure compatibility, the DRAGEN TSO 500 ctDNA v2.6.3 analysis software supports sample sheets containing multiple data sections, with one section per assay (for example, TSO500S_Data and TSO500L_Data).

This section describes how the software validates and processes multi-assay sample sheets and details the specific rules and logic applied during validation.

Planning a Run with a Multi-Assay Flow Cell

1. In the BaseSpace Sequence Hub home page, click "Runs" -> "New Run" -> "Run Planning".

2. In the "Create a Run" page, select "NovaSeq X Series" as instrument platform.

3. Provide information for the first assay until the "Run Review" page.

Analyzing Data from a Multi-Assay Flow Cell

Sample Sheet Validation

The sample sheet validator determines which data section(s) to validate based on the active workflow type. The list of samples to process is generated only from the relevant section (for example, Solid or ctDNA). This ensures that only valid samples for the selected workflow are validated.

The workflow’s data section may contain fewer samples than the BCLConvert_Data section. However, all samples listed in the workflow data section must exist in BCLConvert_Data section. The downstream pipeline will only process samples found in the workflow-specific data section.

Library Prep Kits

Multi-assay sample sheets may include multiple library prep kits values separated by semicolons (";").

Validation Logic:

Adapter Reads

When working with multiple assays, the "AdapterRead" field in the sample sheet may include multiple adapter sequences separated by "+". The adapter read of the selected workflow will be filled in the intermediate sample sheet.

Override Cycles

Override cycles are determined by the index and read length specified in the "Reads" section of the RunInfo.xml file and assay specifics. For multi-assay flow cells, the index and read length provided in RunInfo.xml should be the longer of the sequencing cycles required by the two assays. Override cycles for assays requiring shorter indexes and read lengths will be padded to match the cycle lengths used in the run.

BCL Convert Settings and Data

Starting from BCL:

• Sample Sheet Validator recalculates and inserts the correct override cycles into the BCLConvert_Data section of the intermediate sample sheet.

  • If index lengths vary, the logic has been enhanced to automatically pad shorter indexes with "N" to standardize cycle lengths.

• Sample Sheet Validator also writes in values for: Adapter Reads, MaskShortReads, AdapterBehavior, MinimumTrimmedReadLength. These values are based on the TSO 500 ctDNA library prep kit determined from the input sample sheet.

Starting from FASTQ:

• If users run BCL Convert separately to generate FASTQ files:

  • The input sample sheet must already contain valid override cycles, adapter reads, and other BCL Convert Settings.

  • The resulting TSO 500 ctDNA workflow run will start from FASTQ input files.

BaseSpace Autolaunch

Analyses of samples in multi-assay flow cells can be launched simultaneously from BaseSpace. As long as "Starts from FASTQ" is set to True in Analysis Settings, all samples from a multi-assay flow cell will be demultiplexed first to generate FASTQ files. Then, the ICA pipelines (e.g. DRAGEN TSO 500, DRAGEN TSO 500 ctDNA) will be launched simultaneously to process the samples for each of the assays.

Summary of Rule Changes

[TSO500L_Data] Section

To ensure a successful analysis, follow these guidelines:

1. Avoid any blank lines at the end of the sample sheet; these can cause the analysis to fail.

2. When running local analysis using the command line save the sample sheet in the sequencing run folder with the default name SampleSheet.csv, or choose a different name and specify the path in the command-line options.

ICA with Auto-launch: Sample Sheet Requirements

Refer to the following requirements to create sample sheets for running the analysis on ICA with Auto-launch. For sample sheet requirements common between deployments see . Samples sheets can be created using BaseSpace Run Planning Tool or manually by downloading and editing a sample sheet template

[Cloud_TSO500L_Data] Section

Refer to for this section's requirements.

[Cloud_TSO500L_Settings] Section

[Cloud_Data] Section

[Cloud_Settings] Section

NovaSeq 6000Dx Analysis Application: Sample Sheet Requirements

This section describes fields specific for sample sheets for NovaSeq 6000Dx Analysis Application. For more information on DRAGEN TSO 500 ctDNA Analysis Software sample sheet requirements, refer to the sections above.

[BCLConvert_Settings] Section

DRAGEN TSO 500 ctDNA analysis software is compatible with several instruments and assay workflows, each of which have implications for the sample sheet.

Sample sheet templates contain all required fields, including index sequences in the proper orientation for all indexes from a given library prep kit. The templates are provided as a starting point for creating a sample sheet manually when launching analysis on a standalone DRAGEN server or on ICA using manual launch.

Users can visit the section to learn additional details on required fields and values as they fill in sample information. Use the lookup tables below to select and download the sample sheet template that matches your instrument, assay, and workflow configuration.

v2.6.1-2.6.2

v2.6.3

Instrument Lookup

Use the following InstrumentPlatform values, based on Instrument value, to replace the placeholder value in the above templates:

Prerequisites

The following requirements must be met to install and run DRAGEN TruSight Oncology 500 ctDNA Analysis Application on NovaSeq 6000Dx:

• A NovaSeq 6000Dx sequencing instrument with Illumina Run Manager v1.6.2 installed and paired with DRAGEN Server v4.

• Administrator privileges on Illumina Run Manager.

Installation Instructions

Perform the following steps to download and install the DRAGEN TruSight Oncology 500 ctDNA Analysis Application on NovaSeq 6000Dx installation package:

1. Contact Illumina Customer Care for the link to the installation package. The link expires after 7 days.

2. Download the installation package with the link provided in the email from Illumina Customer Care. The installation package contains the following files:

   1. DRAGEN IRES file: drageninstaller_<DRAGEN_VERSION>.el8.x86_64_prod.ires

External Storage Configuration

Perform the following steps to specify a location to store analysis results:

1. Log into Illumina Run Manager as an administrator.

2. From the Settings menu, select External Storage for Analysis Results.

3. Configure the following parameters:

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.

Fusion Calling

DRAGEN fusion caller performs the fusion calling. The outputs are VCF files. It first scans the genome to discover evidence of possible structural variants (SV) and large indels based on split- and spanning-reads. The evidence is enumerated into a graph with edges that connect all regions of the genome that have a possible breakend association. It then analyzes individual graph edges to discover and score SVs associated with the edges. The substeps of this process include the following items:

• Inference of SV candidates associated with the edge.

• Attempted assembly of the SVs breakends.

• Scoring, genotyping, and filtering of the SV.

• Output to VCF.

Fusion Filtering

In assays, there are hundreds to thousands of fusion candidates in a single sample, all of these candidates are reported in the $SAMPLE_ID.sv.vcf file although Most fusion candidates (~99%) are false positives. The fusion filtering tool, DNA Fusion Filter (DNAFF), distinguishes true fusion calls from the false positives. DNAFF performs the following functions:

• Removes spurious fusions including fusions with only one supporting read pair and fusions that overlap with repeat regions, which are more likely to have sequencing errors.

• Filters nonconfident supporting reads for all fusion candidates based on the following criteria:

  • Filter reads with low-sequence identity with the fusion contig.

Fusion Output Files

Fusion calling results are output in several files:

1. Combined Variant Output File, {SampleID}_CombinedVariantOutput.tsv

2. Fusions CSV, {Sample_ID}_Fusions.csv

3. All Candidate Fusions (with many false positives), {Sample_ID}.sv.vcf

1. Combined Variant Output File

File name: {SampleID}_CombinedVariantOutput.tsv

Fusion calling results are output in the [DNA Fusions] section. Fusion variants must meet the following conditions to be included:

• Passing fusion filtering criteria with "PASS" from DNAFF module.

• Contains at least one gene on the fusion allow list.

• Genes separated by a dash (-) indicate that the fusion directionality could be determined. Genes separated by a slash (/) indicate that the fusion directionality could not be determined.

2. Fusions CSV

File name: {Sample_ID}_Fusions.csv

The fusions file contains all passing fusions identified by the analysis pipeline.

The fusion columns are described in the following table. If you use Microsoft Excel to view this file, genes that are convertible to dates (for example, MARCH1) automatically convert to dd‑mm format (1-Mar).

The following table lists the meaning of the values in the direction column. The values are in the format used by Samtools.

3. Fusion Candidates

All candidates identified through DRAGEN fusion caller performs the fusion calling. The outputs are VCF files.

File name: Logs_Intermediates/DragenCaller/{Sample_ID}/{Sample_ID}.cv.vcf

Overview

The installation script for DRAGEN TruSight Oncology 500 ctDNA Analysis Software installs the following software and dependencies:

1. DRAGEN TruSight Oncology 500 ctDNA Analysis Software itself

2. DRAGEN Software if a compatible version is not present

3. Docker software if a compatible version is not present

4. A script required to generate DRAGEN genome hash table

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

Installation Requirements

Hardware

• DRAGEN server v3 or v4

Software

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

• Docker Software, see table below.

• DRAGEN Software, see table below.

Licenses

• TSO500Combined license

TSO500Combined license has been pre-installed to DRAGEN servers in manufacturing since August 2022. 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.

Permissions

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

• 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 .

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

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

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

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

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

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

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

*DRAGEN Software version

Contrary to the prior versions, the installation scripts for DRAGEN TruSight Oncology 500 ctDNA Analysis Software v2.6.0 and DRAGEN TruSight Oncology 500 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 ctDNA Analysis Software, refer to the respective guide.

Installation Instructions

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

1. Contact Illumina Customer Care at to obtain the DRAGEN TruSight Oncology 500 Analysis Software installer package.

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

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

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

1. Use the following command to update the run script permission: chmod +x /staging/install_DRAGEN_TSO500_CTDNA-2.6.0.run

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

   1. For Docker, use the following command: sudo TMPDIR=/staging /staging/install_DRAGEN_TSO500_CTDNA-2.6.0.run

Running the System Check

After installation is complete, make sure the system functions properly by running the following command: /usr/local/bin/check_DRAGEN_TSO500_CTDNA-2.6.0.sh

The script checks that:

• All required services are running

• Proper Docker image is installed

• DRAGEN TruSight Oncology 500 ctDNA 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_CTDNA_<timestamp>.tgz output file.

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

Uninstall Software

The DRAGEN TruSight Oncology 500 Analysis Software installation includes an uninstall script called uninstall_DRAGEN_TSO500_CTDNA-2.6.0.sh, which is located in /usr/local/bin.

Executing the uninstall script removes the following assets:

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

• Resources found in /staging/illumina/DRAGEN_TSO500_CTDNA

• The dragen_tso500_ctdna Docker image

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

uninstall_DRAGEN_TSO500_CTDNA-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.

DNA expanded metrics are provided for information only. They can be informative for troubleshooting but are provided without explicit specification limits and are not directly used for sample quality control. For additional guidance, contact Illumina Technical Support.

General Troubleshooting on Standalone DRAGEN Server

Sample Sheet Validation Failures

In DRAGEN TruSight Oncology 500 ctDNA Analysis Software, the analysis fails if a sample sheet is invalid. If an invalid sample sheet in suspected, log files can help troubleshoot a failed analysis. Use the following steps to find the log file for the sample sheet:

1. Navigate to the following location /<analysis_output>/Logs_Intermediates/SamplesheetValidation.

2. Open the SamplesheetValidation-.log file

3. Find a line with the following: SampleSheetValidationTask:NA:1 exited with return code 1 which has not been declared as a valid return code.

General troubleshooting for a failed sample sheet:

Valid indexes for assay and index combinations:

Troubleshooting BCL issues:

Troubleshooting FASTQs issues:

Other Troubleshooting Tips

Troubleshooting on ICA

In addition to TSO 500 managed sample sheet validations, ICA managed TSO 500 errors include the following:

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 .

When the analysis run completes, the DRAGEN TruSight Oncology 500 ctDNA 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.

Analysis Output Folder Structure

Single output folder structure is as follows.

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

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

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

Step 1: Run Settings

Step 2: Configuration

Step 3: Sample Settings

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

Step 4: Run Review

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

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

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

Planned Runs Screen (NovaSeq X Series only)

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

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

For more information on run planning, refer to the .

Guided Examples

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

The copy number variant caller in the TSO 500 ctDNA analysis software performs amplification, reference, and deletion calling for 59 genes. The list of genes can be accessed on the .

Target Counts

The first step in the CNV calling algorithm identifies target counts. It includes extracting signals such as read count and improper pairs and putting them into target intervals.

Overview

The installation script for DRAGEN TruSight Oncology 500 ctDNA Analysis Software installs the following software and dependencies:

1. DRAGEN TruSight Oncology 500 ctDNA Analysis Software itself

2. DRAGEN Software if a compatible version is not present

3. Docker software if a compatible version is not present

4. A script required to generate DRAGEN genome hash table

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

Installation Requirements

Hardware

• DRAGEN server v3 or v4

Software

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

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

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

Licenses

• TSO500Combined license

TSO500Combined license has been pre-installed to DRAGEN servers in manufacturing since August 2022. 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.

Permissions

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

• 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 .

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

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

Compatibility with other DRAGEN pipelines

DRAGEN TSO 500 Analysis Software ctDNA v2.6.2 and v2.6.3 (v2.6.2+) are 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 ctDNA pipelines with DRAGEN server software v3.10.19 and above. For more details on DRAGEN multi-version compatibility, please visit .

Software versions without multi-version compatibility referred to as single-version compatible. DRAGEN TSO 500 ctDNA Analysis Software v2.6.2+ will disrupt installations of single-version compatible software from the DRAGEN server. To uninstall a previous version of DRAGEN TSO 500 ctDNA Analysis Software, refer to the respective guide.

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

*DRAGEN TSO 500 Analysis Software ctDNA v2.6.2+ can run on a single server with DRAGEN TSO 500 v2.5.4, and should be installed after v2.5.4. DRAGEN TSO 500 Analysis Software ctDNA v2.6.2+ can be co-installed with multi-version compatible DRAGEN TSO 500 Analysis Software or other DRAGEN pipelines with any order of installation.

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

Installation Instructions

Pre- and Post- Installation Steps

1. Uninstall all existing single-version compatible software on the server (see table above)

2. Install DRAGEN TSO 500 ctDNA Analysis Software v2.6.2+

   • This step will disrupt previously installed single-version compatible software but will not impact multi-version compatible ones (see table above)