The TruSight Oncology 500 product family enables comprehensive genomic profiling of tumor samples, measuring key current and emerging variants and biomarkers. Illumina currently offers three TruSight Oncology 500 (TSO500) products, each with different instrument compatibility and analysis options. Informatics and reporting solutions for the TruSight Oncology products are also available through a partnership between Illumina and Pierian. This bulletin outlines the key differences between each product and the available analysis options, using either local Illumina software or analysis through Pierian Clinical Genomics Workspace (CGW).

The three released TruSight Oncology 500 assays are:

• TruSight Oncology 500 (TSO500)
  Solid tumor assay for FFPE samples with DNA and RNA content. Compatible with NextSeq500/550 and NextSeq 550Dx (in research mode) instruments only. This is the original release from the TruSight Oncology 500 product family.
• TruSight Oncology 500 High-Throughput (TSO500 HT)
  Solid tumor assay for FFPE samples with DNA and RNA content. Compatible with NovaSeq 6000, NextSeq 500/550, and NextSeq 550Dx (in research mode) instruments.
• TruSight Oncology 500 ctDNA (TSO500 ctDNA)
  Liquid biopsy assay for cell-free DNA with no RNA content. Compatible with NovaSeq 6000 instruments only.

The following table provides more details on usage of each assay on each instrument and compatibility with available software analysis pipelines:

Important Notes:

• For TSO500 HT and TSO500 ctDNA NovaSeq runs, use of v1.5 reagent requires version 2.2 of the TSO500 Local App for TSO500 HT and version 1.1 of DRAGEN TSO500 ctDNA Analysis Software. Previous versions of software support only v1 NovaSeq reagents.
• Read length and index length for each assay must be exact to ensure correct processing by the analysis pipelines.
• A manually edited sample sheet is required and available for download from the support sites for both the TSO500 Local App and DRAGEN TSO500 ctDNA Analysis Software. The sample sheet must be saved as a comma-separated text (CSV) file. Illumina recommends setting the system language of the computer used to edit the sample sheet to US English to preserve the correct formatting.
  • TSO500 sample sheet templates
  • TSO500 HT sample sheet templates
  • TSO500 ctDNA sample sheet templates
• The Local Run Manager TSO500 analysis module is compatible only with the on-instrument version of Local Run Manager for the NextSeq (Local Run Manager version 2.4.0) that is bundled with NextSeq Control Software version ≥4.0.1; the module is not compatible with any other version of Local Run Manager, including the off-instrument version.
• TSO500 HT libraries cannot be analyzed with the Local Run Manager TSO500 module.
• Only the TSO500 analysis module for Local Run Manager will generate FASTQs that are compatible as input for the TSO500 analysis pipeline. The Generate FASTQ module for Local Run Manager cannot be used to generate FASTQs for downstream TSO500 analysis as the necessary UMI handling options for the TSO500 libraries are not supported in the Generate FASTQ module.

Compatibility with Pierian CGW

Users of Pierian CGW can interpret results and produce reports from TSO500 data for all three assays in the TSO500 product family. Different file types can be uploaded for analysis depending on the assay type:

* BCL Convert is the preferred software for FASTQ generation compared to bcl2fastq.
** FASTQs created with the Local Run Manager Generate FASTQ analysis module are not compatible with TSO500 analysis and cannot be uploaded to Pierian CGW.